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Valorization of vinasse as broth for biological hydrogenand volatile fatty acids production by means of

anaerobic bacteriaEduardo Bittencourt Sydney

To cite this version:Eduardo Bittencourt Sydney. Valorization of vinasse as broth for biological hydrogen and volatile fattyacids production by means of anaerobic bacteria. Other. Université Blaise Pascal - Clermont-FerrandII, 2013. English. �NNT : 2013CLF22373�. �tel-00914329�

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UNIVERSITE BLAISE PASCAL UNIVERSIDADE FEDERAL DO PARANÁ UNIVERSITE D'AUVERGNE N° d'ordre : D.U : 2373

ECOLE DOCTORALE

SCIENCES DE LA VIE, SANTE, AGRONOMIE, ENVIRONNEMENT N° d'ordre: 613

UNIVERSIDADE FEDERAL DO PARANÁ BIOPROCESS ENGINEERING AND BIOTECHNOLOGY DIVISION

THESIS

Presented by

Eduardo Bittencourt SYDNEY

For the fulfilment of the degree of Doctor of Philosophy

in Process Engineering

VALORIZATION OF VINASSE AS BROTH FOR BIOLOGICAL HYDROGEN AND VOLATILE FATTY ACIDS PRODUCTION BY MEANS OF ANAEROBIC

BACTERIA

M. Soccol Carlos Ricardo, Professeur, Universidade Federal do Paraná Directeur de thèse M. Larroche Christian, Professeur, Université Blaise Pascal, Directeur de thèse M. de Oliveira José Vladimir, Universidade federal de Santa Catarina, Rapporteur et examinateur

M. Bergel Alain, Université de Toulouse, Rapporteur et examinateur

Institut Pascal, UMR CNRS 6602, Axe GePEB – Université Blaise Pascal Laboratory of Bioprocess Engineering – Universidade Federal do Paraná

2

Acknowledgements

I am very grateful to my Brazilian supervisor Prof Carlos Ricardo Soccol for

every professional opportunity given to me during these last 5 years that I had the

pleasure to work alongside.

I would like to thanks my French advisor, Prof Larroche, for the opportunity for

this collaboration work. I’d also like to express my gratitude for the hospitality during

my stay at LGCB.

I take the opportunity to thank Regis Nouaille, now owner of the biotech

enterprise AFYREN, who was my mentor in this topic and became a very good

friend.

I am thankful to thank my wife, Alessandra, a very special person for whom I

have an immeasurable love.

Special thanks to my parents and my brother, who always gave me support

and unconditional love. I don’t think there is a way to express all the love I have for

you.

Thanks to Prof. Dr. Adenise L. Woiciechowski, Prof. Dr. Julio Cesar Carvalho,

Prof. Dr. Luciana P. S. Vandenberghe, Prof. Dr. Michele Rigon Spier, Prof. Dr.

Adriane P. Medeiros, and Prof. Letti for their help. I take the opportunity also to thank

everyone from the LGCB for the friendship and hospitality.

I would also like to thank Ouro Fino Agronegócio, especially Dolivar Coraucci,

Ricardo Asmmann, Carlos Dalmas and Guilherme Moraes. Thanks to all my lab

colleagues from LGCB and LPB.

Thanks CAPES and Fundação Araucária for the financial support.

3

Summary Abstract ....................................................................................................................................... 12

CHAPTER 1

1. Introduction ........................................................................................................................ 15

2 Hydrogen and Volatile Fatty Acids (VFA) Production .......................................................... 16

2.1 Hydrogen Production Processes .................................................................................. 16

2.2 Biotechnological Biohydrogen Production .................................................................. 17

2.2.1 Photo-‐fermentations ........................................................................................... 19

2.2.2 Dark Anaerobic Biohydrogen Production ............................................................ 19

2.2.3 Two phase ........................................................................................................... 20

3 Dark Fermentation Metabolism of Biohydrogen producers ............................................... 20

4 Organic Acids Microbial Production .................................................................................... 25

CHAPTER 2

1 Introduction ........................................................................................................................ 28

1.1 Substrates for Biohydrogen Production ...................................................................... 28

1.1 Sugarcane Vinasse ....................................................................................................... 30

2 Material and Methods ........................................................................................................ 35

2.1 Anaerobic Medium Preparation .................................................................................. 35

2.2 Microorganisms ........................................................................................................... 36

2.3 Biogas Production and Composition Analysis ............................................................. 37

2.4 Ion Chromatography (IC) ............................................................................................. 38

2.5 High Performance Liquid Chromatography (HPLC) ..................................................... 38

2.6 Nuclear Magnetic Resonance ...................................................................................... 39

2.7 Proteins and Aminoacids Quantification ..................................................................... 39

3 Results and Discussion ........................................................................................................ 39

4

3.1 Vinasse Analysis .......................................................................................................... 39

3.2 Effect of carbon source in biohydrogen and VFAs production .................................... 41

4. Conclusion ........................................................................................................................... 46

CHAPTER 3

1 Introduction ........................................................................................................................ 49


2.1 Microorganisms ........................................................................................................... 51

2.2 Medium Composition and Culture Conditions ............................................................ 52

2.3 Culture media and Medium Analysis .......................................................................... 53

2.4 High Performance Liquid Chromatography (HPLC) and Ethanol quantification. ......... 53

2.5 Gas Analysis ................................................................................................................. 54


3.1 Vinasse composition .................................................................................................... 55

3.2 Strains metabolism analysis ........................................................................................ 56

3.2.1 ATCC 8260 ........................................................................................................... 57

3.2.2 ATCC 27021 ......................................................................................................... 58

3.2.3 C6 ......................................................................................................................... 58

3.2.4 VINA ..................................................................................................................... 59

3.2.5 LPB AH1 ............................................................................................................ 61

3.2.6 LPB AH2 ............................................................................................................ 62

3.2.7 LPB AH4 ............................................................................................................ 63

3.2.8 LPB AH5 ............................................................................................................ 64

3.2.9 LPB AH6 ............................................................................................................... 65

3.2.10 LPB AH7 ............................................................................................................... 66

3.3 Conclusions ................................................................................................................. 67

CHAPTER 4

1 Introduction ........................................................................................................................ 70

5


2.1 Medium Composition and Culture Conditions ............................................................ 72


2.3 Gas Measurement and Analysis .................................................................................. 73

2.4 Strains .......................................................................................................................... 73

2.5 Optimization and data analysis ................................................................................... 74


3.1 Consortium LPB AH1 cultivated in vinasse medium supplemented with sugarcane juice. 75

3.2 Consortium LPB AH2 cultivated in vinasse medium supplemented with sugarcane molasses. ................................................................................................................................. 79

4 Conclusions ......................................................................................................................... 82

CHAPTER 5

1 Introduction ........................................................................................................................ 85

2 Material and methods ......................................................................................................... 86

2.1 Culture Conditions and Strains .................................................................................... 86

2.2 Vinasse ........................................................................................................................ 88


2.4 Gas Measurement and analysis ................................................................................... 88

2.5 Other Analysis ............................................................................................................. 89

3 Results ................................................................................................................................. 90

3.1 Biohydrogen and VFAs production in bioreactor scale by the consortium LPB AH1 .. 90

3.1.1 Metabolic analysis ............................................................................................... 92

3.1.2 Hydrogen production potential analysis ............................................................. 93

3.2 Biohydrogen and VFAs production in bioreactor scale by the consortium LPB AH2 .. 94

3.2.1 Metabolic analysis ............................................................................................... 96

3.2.2 Hydrogen production potential analysis ............................................................. 97

4. Conclusion ........................................................................................................................... 98

6

General Conclusion .................................................................................................................. 100

Future works ............................................................................................................................ 102

Bibliographic References .......................................................................................................... 103

Publications in the Field ........................................................................................................... 114

7

List of Figures Figure 1 -‐ A schematic pathway for conversion of renewable to hydrogen via fermentation (53). .. 21

Figure 2 -‐ Metabolic pathway of the production of acids and solvents from glucose in Clostridium acetobutylicum. From Jones and Woods (50). ................................................................................... 22

Figure 3 – Depuration LPB AH2on where vinasse is stored at Usina Catanduva (Catanduva, São Paulo, Brazil). At the day this picture was taken, the flow of vinasse was 350m³.h-‐1. ....................... 31

Figure 4 -‐ Channels for the distribution of vinasse along the field at Usina Catanduva (Catanduva, São Paulo, Brazil. It can also be seen a pump used for vinasse aspersion. ........................................ 32

Figure 5 -‐ Coated channel at Usina Catanduva (Catanduva, São Paulo, Brazil) folowing the new legislation. .......................................................................................................................................... 33

Figure 6 -‐ Technique of producing an anaerobic medium. Boiling under anoxic environment is one important step. .................................................................................................................................. 36

Figure 7 -‐ Biogas quantification using a 60ml syringe. ...................................................................... 37

Figure 8 – The use of a graduated syringe in the quantification of the gas produced during fermentation ...................................................................................................................................... 54

Figure 9 – Graphical 3-‐D and contour displays of the achieved results for optimization of gas production by LPB AH1 consortium cultivated in vinasse medium supplemented with sugarcane juice. ................................................................................................................................................... 77

Figure 10 -‐ Graphical 3-‐D and contour displays of the achieved results for optimization of gas production by LPB AH2 consortium cultivated in vinasse medium supplemented with sugarcane molasses. ............................................................................................................................................ 80

Figure 11 – 2L Bioreactor used in scaled up production of biohydrogen and VFAs by the consortium LPB AH2 (cultivated in vinasse medium supplemented with sugarcane molasses) and LPB AH1 (cultivated in vinasse medium supplemented with sugarcane juice). ............................................... 87

Figure 12 – The system of gas measurement (foreground) adapted to the bioreactor (background).89

Figure 13 -‐ Curves of biomass and VFAs production during the cultivation of the consortium LPB AH1 in vinasse medium supplemented with sugarcane juice. ........................................................... 90

Figure 14 -‐ Biogas production, substrate consumption and pH variation during fermentation of vinasse supplemented with sugarcane juice by the consortium LPB AH1. ........................................ 91

Figure 15 -‐ Representation of the destination of the substrate in terms of VFAs, biomass and CO2 production and cellular maintanance. ............................................................................................... 93

Figure 16 -‐ Biogas production, substrate consumption and pH variation during fermentation of vinasse supplemented with sugarcane molasses by the consortium LPB AH2. ................................. 95

8

Figure 17 -‐ Curves of biomass and VFAs production during the cultivation of the consortium LPB AH1 in vinasse medium supplemented with sugarcane molasses. .................................................... 95

Figure 18 -‐ Representation of the destination of the substrate in terms of VFAs, biomass and CO2 production and cellular maintanance. ............................................................................................... 97

9

List of Tables Table 1 – Most common hydrogen production processes. ................................................................ 17

Table 2 -‐ Overview of biological hydrogen production processes (2). ............................................... 18

Table 3 – Some examples of yields of biohydrogen production achieved using agroindustrial residues as carbon sources. ............................................................................................................... 29

Table 4 -‐ Physico-‐Chemical characterizations of Vinasse (media of 64 samples from 28 ethanol industries from São Paulo – Brazil) (54). ............................................................................................ 34

Table 5 – Mineral composition determined by Ion Chromatography. Those considered following the studies of Neto and Nakahodo, 1995 (54), are signaled with a (*). ............................................. 40

Table 6 – VFAs composition of vinasse by HPLC and RMN analysis. .................................................. 40

Table 7 – Hydrogen production by 5 strains grown in vinasse medium supplemented with different carbon sources. Results represent an average of 20 generation measurements. ............................. 42

Table 8 – VFAs produced (in g/L) by different strains and the consortium VINA in vinasse based medium. ............................................................................................................................................. 44

Table 9 – Hydrogen production by 5 strains grown in Natural Vinasse Media (NVM) supplemented with different carbon sources avoiding hydrogen accumulation. Results represent an average of 20 generation measurements. ................................................................................................................ 44

Table 10 – VFAs concentrations (g/L) at the 4th day of fermentation in vinasse based medium. ..... 45

Table 11 – VFAs concentration (g/L) at the 7th day of fermentation in vinasse based medium. ...... 45

Table 12 – Butyrate/acetate ratio, gas produced and hydrogen content in the gas phase in the experiments carried with each strain. Butyrate/Acetate ratio was considered based on VFAs analysis of the 7th day of fermentation. ............................................................................................. 46

Table 13 – Yields of biohydrogen production of microorganisms grown in pure carbon sources. .... 50

Table 14 -‐ Some yields achieved by using consortia for in fermentation of different substrates. ..... 51

Table 15 – Origin of the samples collected with potential for methane/biohydrogen production. .. 52

Table 16 – Complete composition of the vinasse used during the experiments was carried by BioAgri Laboratory. ............................................................................................................................ 55

Table 17 – Metabolic products of the cultivation of ATCC 8260 in vinasse medium containing different carbon sources. Results include cultivation allowing and avoiding H2 partial pressure. VFAs concentration is shown in g.L-‐1. Results are the average of 5 analyses. ............................................ 57

Table 18 -‐ Metabolic products of the cultivation of the strain C6 in vinasse medium containing different carbon sources. Results include cultivation allowing and avoiding H2 partial pressure. VFAs concentration is shown in g.L-‐1. Results are the average of 5 analyses. ............................................ 58

10

Table 19 -‐ Metabolic products of the cultivation of the consortium VINA in vinasse medium containing different carbon sources. Results include cultivation allowing and avoiding H2 partial pressure. VFAs concentration is shown in g.L-‐1. Results are the average of 5 analyses. .................... 59

Table 20 -‐ Metabolic products of the cultivation of the consortium LPB AH3 in vinasse medium containing different carbon sources. Results include cultivation allowing and avoiding H2 partial pressure. VFAs concentration is shown in g.L-‐1. Results are the average of 5 analyses. .................... 61







Table 27 – Statistical plan used for the optimization of conditions for biohydrogen and VFAs production by the chosen consortia. ................................................................................................. 75

Table 28 – Values of pH and carbon source assigned to each level of the optimization plan. .......... 75

Table 29 – Gas production achieved by cultivating the consortium LPB AH1 under conditions according to the statistical model used for optimization. .................................................................. 76

Table 30 -‐ The equation of the full quadratic model that fit best to the results achieved in this optimization is presented. Coefficient values, standard errors, 95% interval of confidence and T student are also shown. ..................................................................................................................... 77

Table 31 – The ANOVA analysis showed low content of residuals and indicates that the full quadratic equation proposed is valid. ................................................................................................ 78

11

Table 32 – Volatile fatty acids production of the consortium LPB AH1 during optimization. Substrate, succinic, lactic, formic, acetic, propionic and butyric acids are showed in g.L-‐1. .............. 79

Table 33 – Gas production achieved by cultivating the consortium LPB AH1 under conditions according to the statistical model used for optimization. .................................................................. 80

Table 34 -‐ – The equation of the full quadratic model that fit best to the results achieved in this optimization is presented. Coefficient values, standard errors, 95% interval of confidence and T student are also shown. ..................................................................................................................... 81

Table 35 – The ANOVA analysis showed low content of residuals and indicates that the full quadratic equation proposed is valid. ................................................................................................ 81

Table 36 – Volatile fatty acids production of the consortium LPB AH2 during optimization. The concentration of the carbon source, succinic, lactic, formic, acetic, propionic and butyric acids are showed in g.L-‐1. .................................................................................................................................. 82

Table 35 – Composition of the biogas produced during the fermentation by the consortium LPB AH1. .................................................................................................................................................... 92

Table 36 – Composition of the biogas produced during the fermentation by the consortium LPB AH2. .................................................................................................................................................... 96

12

Abstract Vinasse is the liquid waste removed from the base of sugarcane ethanol

distillation columns at a ratio of 12-15 liters per liter of alcohol, resulting in an

estimated production of approx. 350 billion liters in 2012/2013 in Brazil. Vinasse has

a low pH and high chemical oxygen demand, which can cause land desertification

when indiscriminately used as fertilizer. Also, underground water contamination is

being observed in some regions. We evaluated the potential of vinasse as nutrient

source for biohydrogen and volatile fatty acids production by means of anaerobic

consortia. Two different vinasse-based media were proposed, using sugarcane juice

or molasses as carbon source, and were compared to fermentation in a sucrose-

supplemented medium. Pure cultures (4) and consortia (7) were cultured in the

propose media and evaluated for volatile fatty acids (VFAs) and biohydrogen

production. The consortium LPBAH1, originated from faeces of fruit bat, was

selected for fermentation of vinasse supplemented with sugarcane juice and resulted

in a higher H2 yield of 7.14molH2/molsucrose and hydrogen content in biogas of approx.

31% after process optimization. Similarly, the optimized process using the

consortium LPBAH2, originated from a lake of a dairy farm, resulted in 3.66

molH2/molsucrose and 32.7% hydrogen content in biogas. The proposed process is of

great importance for giving a more rational destination to vinasse and expanding

Brazilian energy matrix, reducing the dependence of fossil fuels.

Keywords: biohydrogen, molasses, sugarcane, vinasse, volatile fatty acids,

bioenergy

13

Résumé

La vinasse est le déchet liquide retiré de la base de colonnes de distillation de

l'éthanol de canne à sucre à hauteur de 12 à 15 litres par litre d'alcool, ce qui

entraîne une production estimée à environ 350 milliards de litres en 2012/2013 au

Brésil. La vinasse a un pH faible et une forte demande chimique en oxygène, ce qui

peut provoquer la désertification des terres, si elle est utilisée en excès comme

amendement. En outre, une contamination des eaux souterraines liée aux

épandages est observée dans certaines régions. L'aptitude de la vinasse à jouer le

rôle de source d'éléments nutritifs pour la production de biohydrogène et d'acides

gras volatils par des consortia microbiens anaérobies a été évaluée. Deux différents

milieux à base de vinasse ont été proposés, un avec l’addition de jus de canne à

sucre et l’autre avec l’addition de la mélasse comme source de carbone, et ont été

comparés à un milieu supplémenté en saccharose. Des cultures bactériennes pures

(4) et des consortia microbiens (7) ont été cultivées dans les milieux proposés et la

production des acides gras volatils (AGV) et de biohydrogène ont été evalués. Le

consortium LPBAH1, originaire d’un lac d’une ferme laitière et sélectionné pour la

fermentation de la vinasse avec du jus de canne à sucre, conduit à un rendement en

H2 de 7,14molH2/molsucrose et à une teneur en hydrogène dans le biogaz d'env. 31%

après optimisation. Par ailleurs, le processus optimisé en utilisant le consortium

LPBAH2, originaire de fèces de chauves-souris frugivores, permet d'obtenir 3,66

molH2/molsucrose et 32,7% d'hydrogène dans le biogaz. Le processus proposé est

d'une grande importance pour donner une destination plus rationnelle de la vinasse

et d'élargir le bouquet énergétique brésilien en réduisant sa dépendance des

combustibles fossiles.

Mots-clés: vinasse, biohydrogène, mélasse, canne à sucre, acides gras volatiles,

bioenergie

14

CHAPTER 1

An Introduction to the

metabolism of Biohydrogen and

Volatile Fatty acids of anaerobic

bacteria

15

1. Introduction

Almost 100% of our (increasing) energetic demand is supplied by carbon-

containing fossil sources such as oil, coal and natural gas. The environmental

concerns involving the use of such sources of energy are related to the increase in

atmospheric carbon concentration, which is the main cause of global warming and

climate change.

A reduction of CO2 emissions by more than 50% is recommended by the

Intergovernmental Panel on Climate Change (IPCC) in order to stabilize the CO2

level in the atmosphere at 550 parts per million volume (ppmv) to curb negative

climate effects. In this context the scientific community is doing great efforts to

develop renewable cost-effective sources of energy.

The Framework Convention on Climate Change, sign in Rio de Janeiro in

1992, made global warming a major focus and development of technologies for

reducing/absorbing greenhouse gases (GhG) gained importance. Rubin et al (1992)

divided the greenhouse gases emissions reductions alternatives into three groups:

conservation, direct mitigation and indirect mitigation. Conservation measures

reduced electricity consumption and thus GhG emissions, direct mitigation

techniques capture and remove CO2 emitted by specific emissions sources, and

indirect mitigation involve offsetting actions in which GhG producers support

reductions in GhG emission.

The gradual introduction of fuels with an increasingly lower carbon content per

unit of energy (wood à coal à oil à natural gas) results in a continuous

decarbonisation of the global fuel mix, the main objective of the international

agreement cited before. This chain of lower carbon content fuel ends in Hydrogen.

Hydrogen has a higher gravimetric energy density than any other known fuel and is

compatible with electrochemical and combustion processes for energy conversion

without producing the carbon-based emissions that contribute to environmental

pollution and climate change (36).

16

2 Hydrogen and Volatile Fatty Acids (VFA) Production Anaerobic acidogenesis is known as the first step in the anaerobic digestion of

soluble organic materials to methane and CO2, during which hydrogen is produced.

Because many kinds of bacteria are involved in this process several kinds of organic

acids and alcohols can be produced (70), representing around 55% of the carbon

destination (56).

2.1 Hydrogen Production Processes

Hydrogen does not exist alone in nature. Natural gas contains hydrogen

(about 95% of natural gas is methane, CH4), as does biomass (cellulose), water and

hydrocarbons. The carbon-hydrogen and oxygen-hydrogen bonds present in these

substances, however, have low energy. On the other hand hydrogen-hydrogen

bonds contain much more energy. Methods for producing high-energy content

hydrogen-hydrogen bonds includes a diverse array of primary energy sources such

as wind, solar, geothermal, nuclear and hydropower, can be used to extract

hydrogen from water or other feedstock. This diversity of options enables hydrogen

production almost anywhere in the world.

At present, hydrogen is mainly produced from fossil fuels, either by thermal

and chemical methods (Table 1). About 40% is produced from natural gas, 30% from

heavy oils and naphtha, 18% from coal, and 4% from electrolysis and about 1% is

produced from biomass (51). Nearly 50 million tons of hydrogen is traded annually

worldwide with a growth rate of nearly 10% per year (58).

17

Table 1 – Most common hydrogen production processes.

Method Process Feedstock

Thermal

Steam reformation Natural gas

Thermochemical water splitting Water

Gasification Coal, biomass

Pyrolisis Biomass

Electrochemical Electrolysis Water

Photoelectrochemical Water

Biological

Photobiological Water and algae

Anerobic digestion Biomass

Fermentative microrganisms Biomass

2.2 Biotechnological Biohydrogen Production

Hydrogen obtained from physicochemical methods usually cannot be

regarded as an alternative pollution free energy source. Regarding a sustainable

energy production the biological production of hydrogen represents a particularly

pollution free and energy-saving process, since it is possible to use industrial wastes.

As a consequence it has received special attention by the scientific community

during the last years. Between the years 2000 and 2006 only 391 articles were

published regarding the biological production of hydrogen, while between 2006 and

2012 these numbers were approx. 6 times greater. Only at the first two months of

2013, more than 150 articles in this field were published.

Several processes are currently under development, ranging from biomass

fermentations to photobiological processes. Table 2 gives a more detailed overview

18

of biological hydrogen production processes that are being explored in fundamental

and applied research.

Table 2 -‐ Overview of biological hydrogen production processes (2).

The advantages of the fermentative hydrogen production are the broad

spectrum of applicable substrates as well as high hydrogen production yields (3).

The possibility of coupling the energetic hydrogen production from biomass with the

simultaneous treatment of waste materials is an addition crucial advantage. Both

biohydrogen production and methane from anaerobic digestion are CO2-neutral

since the carbon released by their combustion is derived, directly or indirectly, from

recently fixed atmospheric CO2 (2). Moreover, the emitted carbon associated with

hydrogen produced by microbial fermentation is released during the fermentation

rather than during its utilization, thus potentially allowing easy capture of CO2. Large

scale production will allow recovery of the CO2 for use in microalgae cultures,

greenhouses, storage in chemical form (e.g. as carbonates) or in underground

reservoirs. In this scenario, biological hydrogen production could even be a carbon

negative technology (61). In fuel cells, hydrogen can be converted to electricity very

efficiently, producing only water as a waste product, thus drastically reducing CO2,

NOx, particulate and other emissions that accompany the use of fossil fuels.

As shown in table 2, biohydrogen may be produced biotechnologically by

photo-fermentations, two phase fermentations and dark fermentations. In these

19

cases a specific environment needs to be created in which hydrogen producing

bacteria flourish and others perish (38). Each approach has distinct advantages and

disadvantages with challenging technical barriers to practical application.

The processes for the production of biohydrogen differ primarily concerning

the involved microorganisms, the substrates and the light dependence.

2.2.1 Photo-‐fermentations

Towards the end of the 1930s it was discovered that under certain conditions

unicellular green algae are able to produce hydrogen (4, 5) due to the presence of a

specific enzyme called hydrogenase. Since then hydrogenases and indeed H2

production have been found to be ubiquitous throughout the prokaryotic and

eukaryotic kingdoms.

Physiological studies of cyanobacteria have identified many producing strains,

such as Spirulina platensis (122), Anabaena cylindrica (123), Cycas revoluta (124)

and others. One of the greatest drawbacks of this technology, besides dependence

of light (which influences in bioreactors development, difficulties in large scale

production, among others) is that hydrogen production by cyanobacteria occurs in

such a limiting environment that that cell death is a natural consequence.

Despite that, it can be used as a coupled process to dark anaerobic process.

2.2.2 Dark Anaerobic Biohydrogen Production

Dark hydrogen production is a ubiquitous phenomenon under anoxic or

anaerobic conditions. Anaerobic fermentative bacteria produce hydrogen without

photo energy, and so the cost of hydrogen production is 340 times lower than the

photosynthetic process (6).

A wide variety of bacteria use the reduction of protons to hydrogen to dispose

of reducing equivalents which result from primary metabolism. This oxidation

generates electrons which need to be disposed of for maintaining electrical

neutrality. In aerobic environments, oxygen is reduced and water is the product. In

anaerobic or anoxic environments, other compounds need to act as electron

acceptor, e.g. protons, which are reduced to molecular hydrogen (H2). The capacity

20

to reduce other electron acceptors than oxygen requires the presence of a specific

enzyme system in the micro-organisms: hydrogenases.

2.2.3 Two phase

The idea of two- and multi-stage systems is that the overall conversion

process of the waste stream to biogas is mediated by a sequence of biochemical

reactions which do not necessarily share the same optimal environmental conditions

(52). The principle involves separation of digestion, hydrolysis and acidogenesis

from the acetogenesis and methanogenesis phases.

There are three major advantages to a two-phase design. In a two-phase

system, acid formation is promoted during the acid phase. Therefore the methane

phase is constantly receiving acids to encourage maintenance of high populations of

these methanogen microorganisms. The second advantage is that biomass

production, acidogens and methanogens, can be maintained each at their optimal

growth conditions. The third advantage is higher methane content in the

methanogenic phase reactor (52).

3 Dark Fermentation Metabolism of Biohydrogen

producers

Dark hydrogen fermentation is a ubiquitous phenomenon under anoxic or

anaerobic conditions (i.e., no oxygen present as an electron acceptor). The

advantages of dark fermentation over other processes are: (i) better process

economy for lower energy requirements, (ii) process simplicity, (iii) higher rates of

hydrogen production, and (iv) utilization of low-value waste as raw materials (49).

Figure 1 illustrates the biochemical pathway for conversion of renewable biomass in

to hydrogen via fermentation.

21

Figure 1 -‐ A schematic pathway for conversion of renewable to hydrogen via fermentation (53).

Dark Fermentation is an incomplete oxidation. The profile of the fermentation

products is closely related to biohydrogen yields. In respect to fermentation products,

family Clostridiaceae members include pH-neutral solvent producers, mixed acid,

homoacidogenic and alcohol producers (butyric, acetic and/or lactic acids, ethanol,

propanol or butanol. Among the wide range of by-products of diverse microbial

metabolism, the two pathways producing hydrogen from carbohydrates are

associated with acetate and butyrate. The theoretical yield of H2 per mole of glucose

associated to the production of acetate and butyrate is described in the following

reactions:

C6H12O6 + 2H2O à 2CH3COO- + 2CO2 + 4H2 ΔG’0 = -206 kJ.mol-1

C6H12O6 + 4H2O à 2CH3CH2CH2COO- + 2CO2 + 2H2 ΔG’0 = -264 kJ.mol-1

A maximum of 4 moles of H2 per mole of glucose can be produced concurrently with

the production of energy (206 kJ per mole of glucose) and acetate, which is sufficient

to support microbial growth. The thermodynamical explanation for this limitation is

based on the substrate level, since phosphorylation must produce whole numbers of

ATP and the yield of ATP from glucose must be at least 1 mol/mol for the cell to

survive (11). However, microbial fermentation typically generates more than 1 mol

22

ATP and less than 4 mol H2/mol hexose, quantities that vary according to the

metabolic system and conditions. Figure 2 is a general representation of the

metabolic pathways associated to dark fermentation.

Figure 2 -‐ Metabolic pathway of the production of acids and solvents from glucose in Clostridium

acetobutylicum. From Jones and Woods (50).

The production of hydrogen occurs due to the cleavage of hexoses to

pyruvate through Embden-Meyerhof pathway, with the formation 2 mol of reduced

nicotinamide adenine dinucleotide (NADH). Part of the electrons generated during

23

the oxidation of glucose is involved in the production of butyrate and ethanol, while

the rest is recycled to produce NAD and hydrogen, maintaining the electrical

neutrality.

Three enzymes compete for pyruvate: pyruvate:ferredoxin oxidoreductase

(PFOR), pyruvate:formate lyase (PFL) and the fermentative lactate dehydrogenase

(LDH). The nature of the fermentation depends to a large extent on these enzyme

activities (55). Pyruvate is predominantly cleaved by PFOR to form acetil-CoA, CO2

and reduced ferredoxin (FdH2). Both PFOR and Fd are iron-sulfur proteins which

contains 4Fe-4S clusters. The released H2 yield is dependent upon the fate of

pyruvate, which differs among species due to varying activities of PFL, PFOR and

LDH (12).

This reduced ferredoxin is able to transfer electrons to an iron-containing

hydrogenase which permits the use of protons as a final electron acceptor, resulting

in the production of molecular hydrogen (50). This assures the production of two

moles of hydrogen per mole of glucose consumed. The overall reaction of the

processes can be described as follows:

Pyruvate + CoA + 2Fd(ox) → Acetyl-CoA + 2Fd(red) + CO2

2H+ + Fd(red) → H2 + Fd(ox)

There are two main types of hydrogenases which are phylogenetically distinct

and contain different active sites where the relevant chemistry occurs; Ni–Fe

hydrogenases and [FeFe] hydrogenases. In general, NiFe hydrogenases are poised

to catalyze hydrogen oxidation and [FeFe] hydrogenases are extremely active in

proton reduction. In Clostridia, hydrogen evolution is catalyzed by a soluble [FeFe]

hydrogenase.

The remainder of the hydrogen in the hexose is conserved in the byproduct

acetate and butyrate, and under non-ideal circumstances, more reduced products

like ethanol, lactate or alanine. These reduced products are produced to satisfy

metabolic needs. Acetate allows ATP synthesis, and the reduced products permit the

reoxidation of NADH (which is necessary for continuing glycolysis) (65).

Under abnormal conditions (inhibition of hydrogenase, depletion of iron, for

example), lactate can be produced from pyruvate. This pathway only appears to

24

operate as a less efficient alternative to allow energy generation and the oxidation of

NADH to continue when the mechanisms for the disposal of protons and electrons

by the generation of molecular hydrogen is blocked.

Acetyl-CoA produced by the phosphoroclastic cleavage is the central

intermediate, leading to both acid and solvent production (figure 1). The generation

of hydrogen by fermentative bacteria also accompanies the formation of organic

acids as metabolic products. Highest release of hydrogen is observed when more

oxidized products are produced (acetate and butyrate), which occurs during the

initial growth phase (acidogenic phase). Acid accumulation causes a sharp drop of

culture pH leading to a subsequent inhibition of bacterial hydrogen production; it is

thus required a way to reduce acid production or to neutralize protons outside of the

cells, (53). Inhibition of biohydrogen production can also be caused, and in practice

is the main barrier to achieve high yields, by high H2 partial pressure. According to

the model developed by Ruzicka (1996) (72), as the concentration of dissolved H2

increases in the liquid phase, the transfer of electrons from glucose to H2 becomes

increasingly unfavorable.

During acid-producing metabolism there is a rapid flow of electrons derived

both from the phosphoroclastic cleavage of pyruvate and from NADH to ferredoxin

(50). Since NADH has a higher potential than H2, the dehydrogenation of triose

phosphate to produce 2 mols of H2 can occur only when the partial pressure of H2 is

lower than 6x10-4 atm, while the production of H2 via the oxidation of pyruvate and

ferredoxin can generate another 2 mols of H2 at higher H2 pressure up to 0.3 atm

(71). Thus, in order to obtain H2 yields greater than 2 molH2/molglucose the production

of H2 via triose phosphate dehydrogenation and NADH must be achieved. Since two

moles of NADH are produced during glycolysis, up to a maximum of two additional

molecules of H2 could potentially be generated by NADH pathway.

The formation of relatively reduced organic molecules (e.g. acetate, butyrate)

can inhibit H2 production if these metabolites are allowed to accumulate (12). These

reduced end-products contain additional H atoms that are not liberated as gas (48).

This is the reason why practical production of hydrogen is lower than the theoretic

maximum. For example, the H2 yield from C. butyricum could in theory reach 4 mol

H2/mol hexose although a detailed metabolic analysis of C. butyricum gives a

25

calculation of a maximum of 3.26 mol H2/mol hexose and practical yields obtained

using clostridia rarely exceed 2 mol H2/mol hexose (12).

4 Organic Acids Microbial Production Low-molecular-mass carboxylic acids are important intermediates and

metabolites in biological processes. Known as volatile fatty acids (VFAs) these

homologues and corresponding structural isomers include acetic, propionic, iso- and

n-butyric and iso- and n-valeric acid. The presence of VFAs in a sample matrix is

often indicative of bacterial activity.

Organic acids are some of the end products of anaerobic metabolism to

produce biohydrogen, especially C2 and C4 acids. Generally they are not recovered,

but used in sequential processes as substrate for microbial methane or solvent

production.

If recovered from the broth, organic acids can be produced and sold as

commodity chemicals or further processed into higher value chemicals, biofuels, or

bio-products. Among the acids produced during biohydrogen production are acetic,

butyric, succinic, lactic, formic and propionic acids. Usually, in biohydrogen

processes it is observed a preferential production of acetic and butyric acids.

Considering the economic issues associated to biohydrogen production systems, the

recovery or reuse of such VFAs are of great interest, since H2 production is high.

Butyric acid has many uses in different industries, and currently there is a

great interest in using it as a precursor to biofuels, more specifically biobutanol.

Butyric acid has also applications in the production of low-molecular-weight esters

which have pleasant aromas (perfume industry) or tastes (food flavoring), in animal

feed and in the production of Cellulose Acetate Butyrate (a biopolymer used in high

impact plastics).

Acetic acid is an important feedstock for many chemicals such as vinyl

acetate monomer (for polymers), cellulose acetate, acetic acid esters and acetic

anhydride. Lactic acid is largely used as preservative in food industry (soft drinks,

essence, extracts, fruit juices), as well as propionic acid. Succinic acid is used as

building blocks for chemicals, such as polymers, while formic acid is largely used in

26

leather industry (prevention of mold), in agriculture (silage preservation) and in

animal feed.

27

CHAPTER 2

Preliminary studies on

biohydrogen production in

vinasse-based media by

anaerobic bacteria

28

1 Introduction

1.1 Substrates for Biohydrogen Production

Currently, the cost of H2 generated from biological processes is very high.

Intensive research on biohydrogen is underway, and in the last few years several

novel approaches have been proposed and studied in order to surpass economical

drawbacks that prevent its industrial production (61). Environmental concerns and

evolving legislations on international scale, and considerations about increasing

energy prices, request more participation of net energy producing waste treatment

processes for sustainable pollution control (37). Since the carbon dioxide produced

during the fermentation is derived, directly or indirectly, from recently fixed

atmospheric CO2, the net CO2 charge in dark fermentation processes using

agroindustrial wastes is zero.

In respect to the range of potential substrates which can be utilized by the

broad range of hydrogen producing bacteria it can be stated that, at present, it is

vast and open for further exploration. The major problem in developing large scale

technologies using such wastes is their availability and coverage. In this terms,

domestic and industrial waste waters are good examples, since they will be

produced wherever there is industrial and human activity. The energy accumulated

in wastes can be harvested and converted to hydrogen through dark fermentation.

The energy, now accumulated in hydrogen molecules, can be then converted to

electricity or heat or be stored for further use.

Recently, complex carbon sources, such as molasses (114), food wastes (45),

dairy wastewater (115), mushroom waste (116), rice slurry (116), cheese wey (117),

lignocellulosic materials, glycerol waste (118), vegetable waste (119) and many

others were proved to be susceptible for dark fermentation (Table 3). The more

carbohydrate the wastewater/biomass contains, more suitable it is for biohydrogen

production. Most of times pre-treatment of the complex-carbohydrate source (usually

thermal treatment) is necessary to generate high production rates, otherwise

biohydrogen production is limited by the microorganism(s) hydrolytic activity.

29

Table 3 – Some examples of yields of biohydrogen production achieved using agroindustrial residues as carbon sources.

Microorganism Y(H2/S) (mol.mol-1) Carbon source Reference

Caldicellulosiruptor

saccharolyticus 2.3 bagasse 108

Clostridium butyricum 0.76 Rice straw

hydrolisate 109

Clostridium butyricum 0.75

Sugarcane

bagasse

hydrolisate

110

Clostridium thermocellum 1.47 Delignified

wood fibers 111

Ruminococcus albus 2.59 Sorghum

residues 112

Thermoanaerobacterium

thermosaccharolyticum 2.4

Corn stover

hydrolisate 113

Because of the complex nature of the substrates frequently used and the

often no identification of mixed microbial cultures it is difficult to compare one study

with another (61). The highest H2 yields have been achieved using Clostridia, enteric

bacteria and hyperthermophiles. The strict anaerobic Clostridia are said to produce

hydrogen in higher yields than facultative anaerobes. Extreme thermophiles

achieved yields of approximately 83-100% of the maximal theoretical value of 4

mol/mol (38), but usually grow to low biomass concentrations (resulting in low

production rates). The proper choice of microorganism(s) and substrate is crucial in

the development of a feasible biohydrogen and VFAs production technology.

The use of mixed cultures in the production of hydrogen is an alternative that

is being actively studied by the scientific community. High yields of 2,6 molH2.mol-

1glucose (125) and productivities of up to 150 mmolesH2.L-1.h-1 were described (126).

The main advantages related to mixed culture fermentations are the considerable

low susceptibility to contamination and less toxicity to oxygen, which favor process

handling. Moreover, when complex substrates are used the presence of different

microorganisms generally improves substrate degradation and consequently

30

hydrogen production. On the other side, issues associated to process stability are

noted (modifications on the process or variation on the composition of the substrate

may lead to changes in the microbial community.

1.1 Sugarcane Vinasse

In Brazil, ethanol is produced through a classic fermentation process, in which

yeasts transform sugarcane juice, molasses, or a molasses-juice mixture into

ethanol. This is a biological process that can be represented by the stoichiometric

equation of Gay Lussac:

C12H22O11+ H2O à C6H12O6 + C6H12O6 (a)

C6H12O6 à 2CH3CH2OH + 2CO2 + 23,5 kcal (b)

At the end of the fermentation, practically 100% of the sugar (sucrose) present

in the culture media is consumed by the yeast (usually a Saccharomyces), resulting

in a liquid called wine. The wine has a concentration of ethanol (% in volume)

between 6 and 10°GL, which is recovered by distillation in the top part of distillation

columns, where the present volatile substances are separated based on their

different boiling points.

Vinasse is removed from the base of the distillation columns. It is nothing

more than the fermented broth free of ethanol. It contains some organic solids in

suspension as well as minerals, residual sugar and some volatile compounds.

Considering the ethanol concentration in the wine, vinasse is generated in an

average proportion of 12 to 15 liters for each liter of alcohol produced. According to

Monteiro (33), the physicochemical characteristics of vinasse are: pH 3.8-5.0; Total

solids (g/l) 21.0-85.0; Soluble solids (g/l) 4.0-31.0; Non-soluble solids (g/l) 3.0-13.0;

C.O.D. (mg/l) 15,000-27,000; Water (%) 89-96; Organic matter in total solids (%) 70;

Nitrogen (g/l) 1.0-3.5; Phosphorus (g/l) 0.4-4.0; Potassium (g/l) 9.0-13.0; Magnesium

(g/l) 0.8-1.5; but this varies considerably and should be analyzed case by case.

Because of its production rate and its chemical characteristics vinasse constitutes

the largest pollution source of the Brazilian ethanol industry.

31

Currently, the destination given to vinasse is its aspersion over sugarcane

plantations. Vinasse is usually stored in depuration lagoons (Figure 3) prior use.

Channels are built through sugarcane plantations where vinasse drains and a motor

pump truck is responsible to sprinkle the liquid (Figure 4). Its application as fertilizer

has some advantages, especially in terms of productivity, but the amount used might

be well determined. There is a maximum rate of vinasse application in the field,

based on soil composition (but in practice soil characterization is not carried and

inspection by environmental organizations is very difficult to be handled).

Figure 3 – Depuration LPB AH2on where vinasse is stored at Usina Catanduva (Catanduva, São Paulo, Brazil). At

the day this picture was taken, the flow of vinasse was 350m³.h-‐1.

32

Figure 4 -‐ Channels for the distribution of vinasse along the field at Usina Catanduva (Catanduva, São Paulo,

Brazil. It can also be seen a pump used for vinasse aspersion.

.

When used in excess, productivity reduction, late maturation and low sucrose

content are commonly observed (120). When vinasse is produced in excess and

cannot be used as fertilizer, which is very common, industries throw it in areas called

“sacrifice zones”. In this area the soil becomes very salty and acid causing

desertification and rendering it unusable for any other purpose. In long-term these

characteristics are also noted in productive land, causing productivity decrease, late

maturing and decrease in sucrose content (120). In 1986 40% of the vinasse

produced in Brazil was not used as fertilizer and was thrown in sacrifice zones (121).

Unfortunately no updated data collection is available (informal conversations with the

environmental manager of an industry in São Paulo indicates that this number is

approx. 25%).

Seiju Hassuda (34) identified infiltration problems due to vinasse aspersion in

Bauru Aquifer (SP-Brazil), the most important aquifer in Brazil. This problem is not

only related to the sacrifice zones, since it can be seen in the Figure 4 that no

protection is given to avoid vinasse infiltration in the soil. New government

regulations are now forcing the industries to coat the channels (Figure 5), but

inspection is very limited. Mellissa et al (35) stated vinasse can promote changes of

soil physical properties in two different ways: (i) improving aggregation, consequently

33

raising the capacity of infiltration of water in the soil, thus causing ions leaching and

contamination of the groundwater; and (ii) promoting the dispersion of soil particles,

reducing the rate of infiltration and increasing the runoff, resulting in possible

contamination of surface water.

Figure 5 -‐ Coated channel at Usina Catanduva (Catanduva, São Paulo, Brazil) folowing the new legislation.

In this context, it is of great importance to give a more rational destination to

vinasse or at least reduce its toxicity.

During the last decades, ethanol production has increased very rapidly. Brazil

is, nowadays, the second higher ethanol producer in the world. Recent international

incentive and demand for biofuels production influenced Brazilian ethanol industries,

increasing production. Thus, the problem of vinasse disposal will worsen. Indeed, its

continuous discharge onto land can endanger the chemical and physical structure of

the soil, reduce yields and lead to serious groundwater pollution problems.

Usina Sao Martino (Sao Paulo – Brazil) installed a pilot plant for the

biodigestion of vinasse, obtaining biogas, which is used to burn as fuel in the boilers

of the plant. The technology has reached a reasonable degree of maturity due to the

successive experiments, but some uncertainties decelerated its scale up (42). In the

year 2012 a 612 MWh biogas plant was installed at Companhia Alcoolquímica

Nacional (Vitória do Santo Antão, Pernambuco, Brazil) for the processing of 20% of

the vinasse produced daily.

Regarding the composition depicted in Table 4, vinasse is an interesting

substrate for microorganism growth because it presents a great amount of

micronutrients. Iron, magnesium, phosphorus and nitrogen content are interesting for

34

the development of biohydrogen production. The fact that some successful cases of

methane production are described also reinforced the possibility of hydrogen

production.

Table 4 -‐ Physico-‐Chemical characterizations of Vinasse (media of 64 samples from 28 ethanol industries from São Paulo – Brazil) (54).

Parameter Unity Medium Value

pH 4,15

Brix ºB 18,65

DBO5 mg/L O2 16494,76

DQO mg/L O2 28450,00

Calcium mg/L CaO 515,25

Chloride mg/L Cl 1218,91

Cooper mg/L CuO 1,20

Iron mg/L Fe2O3 25,17

Phosphorus mg/L P2O4 60,41

Magnesium mg/L MgO 225,64

Manganese mg/L MnO 4,82

Nitrogen mg/L N 356,63

Ammonia Nitrogen mg/L N 10,94

Potassium mg/L K2O 2034,89

Sodium mg/L Na 51,55

Sulfate mg/L SO4 1537,66

Sulfite mg/L SO4 35,90

Zinc mg/L ZnO 1,70

Ethanol- CG mL/L 0,88

Glycerol mL/L 5,89

Because low amounts of fermentable carbon are present in its composition,

vinasse might be enriched with a carbohydrate source to allow the production of

great quantities of hydrogen. Some cheap fermentable carbon sources are available

in Brazil, especially in the ethanol industries, where vinasse is generated: sugarcane

35

molasses and sugarcane juice. Molasses arises from sugar production, after the

sugarcane juice concentration and centrifugation. Usually it is used in yeast

fermentation for ethanol production, together with sugarcane juice.

Considering the usage of molasses or sugarcane juice as carbon sources

they do not burdens on the cost of the medium for biohydrogen production. At this

point, promotion and maintenance of anaerobic environment are the processes that

will probably impact most significantly the price of the final product. If purified,

biohydrogen can be used in chemical industry or in fuel cells for the production of

electricity. Otherwise, the hydrogen-rich biogas can be used for heat generation

through direct combustion or in boilers.

Preliminary studies on the evaluation of using vinasse as culture medium for

biohydrogen and VFAs production by anaerobic bacteria were carried at the

Laboratoire de Gènie Chimique et Biochimique (LGCB) at the Université Blaise

Pascal - Clermont-Ferrand, France, and are described in this chapter.

2 Material and Methods

2.1 Anaerobic Medium Preparation The procedures for promoting an anaerobic culture were based on the

technique developed by Ralph S. Wolfe during the mid-1970s, which is generically

referred to as “the Balch technique”.

The removal of oxygen and lowering the redox potential of culture media by

the addition of a reducing agent are the two crucial parts of the technique. The

removal of oxygen was achieved by boiling the medium under an anoxic ambient

(CO2 atmosphere) (Figure 6). The CO2 was scrubbed free of oxygen in a heavy-

walled copper tube packed with copper turnings and heated to 150–200ºC in a tube

furnace.

36

Figure 6 -‐ Technique of producing an anaerobic medium. Boiling under anoxic environment is one important

step.

Bicarbonate was added at the temperature of 85ºC and Cysteine-HCl at 65ºC

as reducing agents to lower the redox potential of medium. To assure oxygen

removal Resazurin was used as indicator. After naturally cooling to room

temperature the medium was distributed into 15ml Hungate tubes under pure CO2

atmosphere and autoclaved.

The experiments were carried out in 15 ml Hungate tubes, with working

volume of 6 ml, sealed with autoclavable Bakelite lids with rubber stopper and

incubated in a shaker at 37ºC and 30 rpm.

Fermentation medium was constituted by pure vinasse supplemented with

10g/L of one of the following carbon sources: glycerol, sucrose and glucose. The

cultures were maintained at these conditions for 1 week and then inoculated in a

new medium. Each new culture will be called “generation”.

2.2 Microorganisms Two known Clostridium strains, C. saccharoperbutylacetonicum and C.

beijerinckii purchased from ATCC (ATCC #27021 and #8260, respectively), two

isolated Clostridium strains (C2 and C6) and one natural vinasse consortium (VINA)

were used.

37

The two ATCC strains are potential hydrogen and VFAs producers able to use

sucrose as carbon source. The isolated Clostridium strains, C2 and C6, were chosen

based in hydrogen and VFAs production among other isolated strains from the

Laboratory Génie Chimique et Biochimique (LGCB). C6 is capable of using sucrose

as carbon source, while C2 can only growth in glucose medium. The vinasse natural

consortium, VINA, was obtained directly by incubating anaerobic pure vinasse

supplemented with sucrose.

2.3 Biogas Production and Composition Analysis

Biogas production in Hungate tubes cultures was periodically measured using

60 mL plastic syringes (Figure 7). Gas production was measured and analyzed twice

in a week or daily, according to the experiment. Those cultures degassed daily were

considered free of H2 partial pressure. Hydrogen total production and production rate

was calculated based on the volume of medium, gas composition and intervals of

analysis.

Figure 7 -‐ Biogas quantification using a 60ml syringe.

The biogas sampled from the headspace was analyzed using a MicroGC

Agilent 300A with 2 channels for gas analysis. Hydrogen (H2), oxygen (O2), nitrogen

(N2) and methane (CH4) was measured through a MoleSieve 5A (10mx0.32mm)

column operated at 100ºC, at injector temperature of 95 °C, using argon as the

38

carrier gas at 30ψ. Carbon dioxide (CO2), hydrogen sulfite (H2S), air and water vapor

(H2O(v)) were measured in a PLOT U (8mx0.32mm) column operated at 70ºC, at

injector temperature of 70 °C, using hydrogen as carrier gas at 15ψ. Each column

was connected to a separated TCD for detection.

2.4 Ion Chromatography (IC)

Ion chromatography (761 Compact IC 817 Bioscan chromatograph) was used

for the determination of vinasse mineral composition. For cations analysis a Metrohm

METROSEP C3 250/4.0 (250 mL x 4.0 mmID) column was used. Analytical

conditions were: 3.5 mM HNO3, 1.0 mL/min, 40ºC, 20 µL sample volume, 11.2 MPa.

A standard chromatogram was prepared with the following cations: Ca, Mg, K, Na,

Zn, NH4 and Fe. Anions analyses were made in a Metrosept A Supp 5 250/4.0

column (250 mL x 4.0mmID). Analytical conditions were: 3,2mM Na2CO3 + 1mM

NaHCO3, 1.0 mL/min, 40ºC, 20 µL sample volume, 10.2 MPa. A standard

chromatogram was prepared with the following anions: F, Br, NO3, PO4, SO4 and Cl.

All reagents used were analytical grade (Sigma–Aldrich).

2.5 High Performance Liquid Chromatography (HPLC)

Organic components were determined through High Performance Liquid

Chromatography (HPLC). Before injection the samples (2 ml) was treated with 0.25

ml of BaOH (0.3M) and 0.25 ml of ZnSO4 (5%), centrifuged for 10 min at 104xg and

filtered (Milipore 0,2µm), to avoid column obstruction by suspended solids.

The HPLC equipment used was an Agilent 1100, equipped with 2 ion

exclusion columns (Phenomenex Rezex ROA 300 x 7.8 nm) placed in series in a

50ºC oven. A 2mM sulfuric acid in ultrapure water solution (Millipore, MilliQ plus) was

used for elution at 0.7 ml flux (pomp HP 1100 series, Agilent Technologies). The

chromatograph is equipped with an automatic injector (Agilent Rhéodyne). Detection

was done through a refractive index detector (HP 1100 series) and the signals

integrated (HP 1100 series). The acquisition is done by the HPChem program

(Agilent Technologies). The compounds quantified by this method are cellobiose,

39

glucose, fructose, succinate, lactate, formate, acetate, propionate, isobutyrate,

butyrate, isovalerate and valerate.

2.6 Nuclear Magnetic Resonance

Measurements of NMR spectra were performed at 27 °C on a 300 or 500 MHz

Avance Bruker spectrometer equipped with 5mm TXI 1H, 13C, 15N probe with inverse

detection.

Samples were centrifuged (10000 rpm, 10min) and to 540 µl of supernatant,

60 µl of a solution TSPD4 (2,08 ml TSPD4 10mM + 7,92ml D2O - used as internal

reference for chemical shift and quantification).

2.7 Proteins and Aminoacids Quantification

Proteins were quantified by the method of Bradford. The Dye stock was

prepared by dissolving 100 mg of Coomassie Blue G in 50 ml of methanol, followed

by the addition of 100 ml of 85% H3PO4 and dilution to 200 ml with distilled water.

Due to the natural color of vinasse, the methodology was adapted. The procedure

was made by adding 1 ml of dye stock to 4 ml of sample. The absorbance was read

at 595 nm. A standard curve was made using vinasse instead of water by adding

known quantities of BSA to each sample, in order to minimize the effect of vinasse’s

color on the results. The amount of protein in vinasse was determined based on the

equation obtained by the linearized curve.

3 Results and Discussion

3.1 Vinasse Analysis

The mineral composition of vinasse was analyzed by ion chromatography and

is presented in Table 5. The ions that could not be determined were considered

based on the analysis made by Neto and Nakahodo in 1995 (54). The organic

composition of vinasse was determined by HPLC and RMN (Table 6).

40

Table 5 – Mineral composition determined by Ion Chromatography. Those considered following the studies of Neto and Nakahodo, 1995 (54), are signaled with a (*).

Mineral mg/L

Ca 515,25

Cl 1218,91

P 120,82

Mg 244,71

N 356,63

K 1750,9

Na 51,55

SO4 1537,66

NNH3* 10,94

Cu* 1,2

Fe* 25,17

Mn* 4,82

SO3* 35,9

Zn* 1,7

Table 6 – VFAs composition of vinasse by HPLC and RMN analysis.

VFA mg/L Ethanol 0 Butyrate 1300

Propionate 1100 Acetate 700 Lactate 200

Proteins were quantified by the method of Bradford and resulted in

approximately 670 mg/L. Aminoacids quantified by the ninhydrin method resulted in

470 mg/L. Since no carbohydrate was detected, it was expected the necessity to

supplement vinasse medium with an organic source of carbon for feasible

biohydrogen production.

As expected, vinasse analysis indicated that it is a rich residue, containing a

great variety of mineral compounds. This is interesting for bacterial growth and also

41

in promoting hydrogen production (especially iron). The presence of some VFAs is

not ideal but they are present in low amounts and might not be a problem for

biohydrogen production.

3.2 Effect of carbon source in biohydrogen and VFAs production

Since vinasse analysis indicated absence of sugars, different carbon sources

were added to vinasse. The choice of the carbon source to be added is of great

economic importance to the process. The use of pure carbon sources in these

preliminary experiments was carried in order to evaluate the metabolism and the

potential of each strain prior to the use of complex substrates.

The following substrates were evaluated in these preliminary experiments:

(i) Sucrose: sucrose is present in high concentrations in sugarcane

molasses, a residue from industrial sugar production, and also in sugarcane juice,

which is extracted for both alcohol and sugar production. Because of its availability

sucrose (or alternative sources of sucrose) is probably the most interesting carbon

source to be used.

(ii) Glycerol: glycerol is another interesting carbon source because it is

produced in great amounts in biodiesel industries, which are largely increasing in the

last years. The fate of the glycerol generated in biodiesel industries is object of great

concern due to the enormous amounts produced, making it an interesting substrate

for the process proposed in this work.

(iii) Glucose was also tested to serve as model as it is the most easily

assimilated source of carbon by the majority of microorganisms. It can be obtained

from complex substrates through hydrolysis.

3.2.1.1 Hydrogen production in vinasse medium supplemented

with pure carbon sources

During 20 generations the gas produced during fermentation was measured

and analyzed twice a week (4th and 7th days of fermentation). Results of average

hydrogen production rate (in mL.L-1.day-1) and average total production (in mLH2.L-1)

of each strain are showed in Table 7.

42

Table 7 – Hydrogen production by 5 strains grown in vinasse medium supplemented with different carbon

sources. Results represent an average of 20 generation measurements.

Strain Carbon Source H2 (ml/L/day) Total H2 (ml/L) Hydrogen in

Gas Phase (%)

C2 Glucose 104.0±46.5 728 10

C2 Glycerol 7.9±1.9 55.3 3

C2 Sucrose 0 0 0

C6 Glucose 237.6 1663.2 9

C6 Glycerol 7.9±2.7 55.3 3

C6 Sucrose 197.3±11.5 1381.1 13

VINA Glucose 643.4 4503.8 25

VINA Glycerol 20.0±7.0 140.0 2.5

VINA Sucrose 262.6±66 1838.2 12

ATCC 27021 Glucose 730.5 5113.5 35

ATCC 27021 Glycerol 0 0 0

ATCC 27021 Sucrose 587.8±160 4114.6 36

ATCC 8260 Glucose 780.3 5462.1 40

ATCC 8260 Glycerol 0 0 0

ATCC 8260 Sucrose 635.3±89 4447.1 34

It is can be observed that the pure strain C2 was not capable of growing in

sucrose vinasse medium, while ATCC 8260 and ATCC 27021 were not capable of

growing in medium supplemented with glycerol.

Hydrogen production was higher in vinasse medium supplemented with

glucose for all the strains tested. The lower yields were achieved when glycerol was

used as carbon source. For all strains, a fluctuation in H2 production was observed,

which might be a consequence of the high complexity of natural vinasse.

The consortium VINA presented a great difference in terms of hydrogen

production when grown in glucose and sucrose medium. This indicates that this

consortium is composed by some microorganisms which cannot use sucrose or

fructose as carbon source to produce hydrogen.

The pure strains ATCC 27021 and ATCC 8260 presented the best results for

biohydrogen production. The higher volume of hydrogen produced were

43

accompanied by higher hydrogen concentration on the gas phase, which is also

important for future gas purification processes.

In those media supplemented with sucrose, H2 production was considerable

high and not much lower than when glucose was used, except for VINA consortium.

An interesting point that might be considered is the availability of cheap sucrose

sources in Brazilian Ethanol Industries (molasses and sugarcane juice). For these

reasons sucrose was chosen as the carbon source for the following experiments.

3.2.1.2 Liquid phase analysis of cultures carried in vinasse medium

with sucrose as carbon source

In the 7th day of the cultures carried in sucrose supplemented vinasse medium

samples were withdrawed and analyzed. Results of HPLC and RMN analysis of the

fermented broth are presented in Table 8.

Acetate and butyrate were the main VFA products by VINA, C2 and C6.

These strains also produced ethanol and propionate. ATCC 27021 and ATCC 8260

presented acetate, butyrate and propionate as main products. Lactate was also

produced in significant amount, suggesting that the metabolism of Acetyl Co-A (and

consequently H2 and VFAs) was blocked. Valerate and isobutyrate were found in

trace concentrations and are not showed. The presence of more reduced products,

such as ethanol, is an evidence of a metabolic shift caused by hydrogen partial

pressure caused by non-continuous gas measurements.

.

44

Table 9 – VFAs produced (in g/L) by different strains and the consortium VINA in vinasse based

medium.

Strain Acetate Formate Butyrate Ethanol Propionate Lactate

ATCC 27021

1.79±0.25 0 3.53±0.14 0 1.42±0.0 0.79±0.09

ATCC 8260

1.64±0,11 0 4.28±0.21 0 1.39±0.05 0.59±0.0

VINA 1.7±0.2 0.6±0.2 2.3±0.31 1.8 0.9±0.0 0.25±0.05

C2 1.3±0.0 0.15±0.05 1.6±0.09 2.4 1.4±0.3 0.1

C6 1.4±0.2 0.5±0.3 1.3±0.11 2.6±0.11 1.0±0.1 0.2±0.15

3.2.1.3 Metabolism effect when minimizing H2 partial pressure

Knowing that hydrogen partial pressure is central in hydrogen production an

experiment was carried withdrawing (and analyzing) daily the gas produced

during fermentation. Results are showed in Table 10.

Table 10 – Hydrogen production by 5 strains grown in Natural Vinasse Media (NVM) supplemented with different carbon sources avoiding hydrogen accumulation. Results represent an average of 20 generation

measurements.

Strain Carbon Source H2 (ml/L/day) Total H2 (ml/L) Hydrogen in

Gas Phase (%)

C2 Glucose 100.0 700.0 7

C6 Sucrose 202.8 1419.6 10

VINA Sucrose 403.9 2827.3 13.4

ATCC 27021 Sucrose 2526.3 17684.1 33

ATCC 8260 Sucrose 1895.8 13270.6 24

A great increase of hydrogen production was observed for both ATCC strains

and for the consortium VINA in comparison with the experiments described in the

previous section (Table 7).

45

Since gas production was greatly increased by minimizing H2 partial pressure,

analysis of the liquid phase was also carried. The VFAs analysis at the 4th day of

cultivation is showed in Table 11. At this point no sugars were detected in

cultivations of C6, C2 and VINA, while in cultivations of ATCC 27021 and ATCC

8260 3.5g/L and 0.37g/L, respectively, were detected. The negative concentrations

found for propionate and lactate indicates the consumption of these metabolites in

comparison to the non-fermented medium.

Table 11 – VFAs concentrations (g/L) at the 4th day of fermentation in vinasse based medium.

Strain Acetate Formate Butyrate Ethanol Propionate Lactate Succinate

ATCC 27021

0.90 0 2.49 0 -0.38 -0.37 0.16

ATCC 8260

0.98 0 3.13 0.08 -1.10 -0.37 0.16

VINA 1.05 0.4 0.41 2.60 -0.70 -0.37 0

C2 0.82 0.32 0.49 2.00 -0.38 -0.37 0

C6 0.98 0.4 0.17 2.64 -0.70 0 0.08

Another VFAs analysis was carried in the last day of culture (7th day), when

the substrate was completely consumed in all cultures. Results are showed in Table

12.

Table 12 – VFAs concentration (g/L) at the 7th day of fermentation in vinasse based medium.

Strain Acetate Formate Butyrate Ethanol Propionate Lactate Succinate

ATCC

27021 1.14 0 3.61 0 -1.10 -0.37 0.16

ATCC 8260

1.94 0.08 2.55 0.16 -1.10 -0.37 0.24

VINA 0.98 0.40 0.41 2.48 -0.70 -0.37 0

C2 0.98 0.4 0.17 2.60 -0.70 0 0.08

C6 1.14 0.48 0.25 2.88 -0.62 -0.13 0

46

Strains ATCC 27021 and ATCC 8260 produced acetate and butyrate as main

VFAs, while C2, C6 and the consortium VINA produced mostly acetate and ethanol.

All strains presented the capacity of consuming lactate and propionate under the

conditions tested.

Comparing the VFAs production in 4th and 7th day it can be observed a

considerable increase in butyrate concentration in ATCC 27021 and in C2 (although

butyrate concentration in C2 is low) and in acetate for ATCC 8260. The other VFAs

didn’t show considerable variation, which is compatible with the presence of sugar in

4th day and with the consumption of propionate between the 4th and 7th day.

It is also interesting to note the effect of the H2 partial pressure in VFAs

production by comparing tables 8 and 11. When H2 partial pressure was minimized it

was observed an enhanced production of more oxidized products.

Based on VFAs and gas analysis it was noted that high butyrate/acetate ratio

is related to higher hydrogen content in the gas phase (Table 13). At the same time

there is a relation between the amount of gas produced and the hydrogen

concentration in the gaseous phase.

Table 13 – Butyrate/acetate ratio, gas produced and hydrogen content in the gas phase in the experiments carried with each strain. Butyrate/Acetate ratio was considered based on VFAs analysis of the 7th day of

fermentation.

C2 C6 VINA ATCC 8260 ATCC 27021

Butyrate/Acetate 0,17 0,22 0,42 1,31 3,17

H2 (%) 7 10 13,4 24 33

Gas (Lgas/Lmedium) 10 14,19 21,10 55,29 53,59

4. Conclusion Vinasse has proved to be an interesting base medium for biohydrogen and

VFAs production by anaerobic bacteria. Higher yields were achieved when

glucose was used as substrate, closely followed by the yields achieved in

sucrose vinasse medium. Due to process economics (higher availability and no

47

need of processing) sucrose was chosen as the substrate for VFAs biohydrogen

production in vinasse based medium.

48

CHAPTER 3

Metabolic analysis of potential

strains and consortia for the

production of biohydrogen and

VFAs in vinasse medium

49

1 Introduction The anaerobic degradation of organic matter by heterotrophic microorganisms

can liberate H2 at high rates, depending on the particular organisms and conditions.

Hydrogen producing microorganisms can be divided in four groups: strict anaerobes

(I), facultative aerobes (II), aerobes (III), co- and mixed cultures (IV) (2). The first

group is the most studied, and the most important microorganisms are Clostridia,

Rumen bacteria, thermophiles and methanogens. Known facultative anaerobes

include Enterobacter, E. coli and Citrobacter and known aerobes are Alcaligenes

and Bacillus.

Most studies described in the literature use glucose and sucrose as carbon

sources for biohydrogen production via dark fermentation (

Table 14). The search for endogenous microorganisms in the development of

bioprocess technologies is of great importance due to their increased adaptation to

specific conditions. It is believed that for future applications of biohydrogen

processes the use of mixed cultures from industrial wastes might have more

advantages because they are less susceptible to contamination by H2-consuming

bacteria and are more sensitive to process variations.

Other advantages of making use of a diverse microbial community when

(agro)industrial wastes are used as substrate are: (i) development of a food web

where specific groups of organisms maintain low concentration of critical

intermediate products and promote flux of carbon and electrons from the feedstock

material to the desired end product by reducing direct inhibition of microbial activity

by metabolic intermediates (121); (ii) higher adaptation to substrate variation, which

is an intrinsic characteristic of (agro)industrial wastewaters, due to the presence of

alternative metabolic pathways.

Microflora from various sources has been used as inoculum for hydrogen

production (see some examples in

50

Table 15). Digester sludge from the treatment of urban wastewater, livestock waste,

anaerobic sludge effluent and soil are common sources of inoculum. To avoid

methane producers, heating (75-121oC for 15-120min) or chemical (2-

bromoethanesulfonate) treatments are frequently used. Table 14 – Yields of biohydrogen production of microorganisms grown in pure carbon sources.

Microorganism Y(H2/S)

(mol.mol-1)

Carbon

source Reference

Clostridium acetobutylicum 1.39 glucose 93

Clostidium beijericnkii 1.86 glucose 94

Clostidium beijericnkii 4.20 sucrose 95

Clostidium butyricum 1.35 sucrose 96

Clostidium

saccharoperbutylacetonicum 1.72 glucose 97

Clostridium sp. 3.24 lactose 98

Clostridium beijericnkii 3.9 cellobiose 95

Clostridium butyricum 0.72 xylose 99

Enterobacter aerogenes 1.89 sucrose 100

Enterobacter aerogenes 0.83 lactose 100

Enterobacter aerogenes 0.39 fructose 101

Escherichia coli 1.95 glucose 102

Klebsiella oxytoca 1.5 sucrose 103

Ruminococcus albus 2.11 glucose 104

Ruminococcus albus 1.44 arabinose 105

Thermoanaerobacterium

thermosaccharolyticum 7.44 lactose 106

Thermoanaerobium

thermosaccharolyticum 2.42 glucose 107

51

Table 15 -‐ Some yields achieved by using consortia for in fermentation of different substrates.

Culture Y(H2/S) (mol.mol-1) Carbon source Reference

Anaerobic Digester 2.18 Glucose 87

Rice Rhizosphere microflora 2.3 Apple pomace

wastes 88

Activated and Digested

sludge 1.16 Glucose 89

Digested wastewater sludge 6.12 Sucrose 90

Methanogenic granules 1.2 Glucose 91

Anaerobic mixed culture 5.15 Sugar-beet pulp 92

Since the choice of the microorganism is of great importance, this chapter

reports the search and evaluation of potential strains and consortia from the Brazilian

environment for the production of biohydrogen and VFAs. Samples were taken from

environments capable of supporting anaerobic forms of life. The metabolic behavior

of each strain/consortium was evaluated in vinasse medium supplemented with

cheap sources of sucrose (sugarcane juice and sugarcane molasses) and under the

presence/absence of hydrogen partial pressure.


2.1 Microorganisms

Besides the strains ATCC 8260, ATCC 27021, C2, C6 and the consortium

VINA used in the experiments described in the previous chapter, 9 samples of

Brazilian environments with proper conditions for the development of methane

producers (and consequently, hydrogen producers) were collected. The name of the

strains and origin are described in Table 16.

52

Table 16 – Origin of the samples collected with potential for methane/biohydrogen production.

Name Origin

LPB AH1 Faeces from fruit bat (unknow species)

LPB AH2 Lake of a dairy farm

LPB AH3 Soil used for Sugarcane cultivation

LPB AH4 Domestic sewage

LPB AH5 Swine faeces

LPB AH6 Mangrove from Matinhos-Paraná

LPB AH7 Cow feaces

LPB AH7 Puddle in a cave at São Paulo

2.2 Medium Composition and Culture Conditions


volume of 6 ml, sealed with autoclavable Bakelite lids with rubber stopper and

incubated in a shaker at 37ºC and 30 rpm. The cultures were maintained at these

conditions for 1 week and then inoculated in a new medium. 1 ml of culture was,

then, inoculated in 5 ml of medium. Each new culture will be called “generation”.

Anaerobic environment and medium was carried according to the Balch

technique. Bicarbonate was added at 85ºC and Cysteine-HCl at 65ºC as reducing

agents to lower the redox potential of medium. Otherwise stated, medium pH was

adjusted to 6.8 with 1N KOH.

Anaerobic media containing vinasse and different sucrose sources were used:

i) Sucrose + vinasse, ii) Sugarcane molasses + vinasse and iii) Sugarcane juice +

vinasse. Carbon source concentration in the media was fixed in 10g/L. Sugarcane

molasses addition to reach 10g/L was based on obrix while sugarcane juice sugar

content was quantified by the phenol sulphuric method. All strains and consortia

were cultivated in this media during 15 generations before analysis.

53

2.3 Culture media and Medium Analysis

Because the vinasse used at the preliminary studies (presented in chapter 2)

was concentrated and then reconstituted prior to use, a new fresh vinasse was used

in media preparation. This decision was based on the unknown effects of

concentration in vinasse composition and the necessity to carry the experiments

(described in chapters 3, 4 and 5) with the same vinasse.

The new vinasse was a courtesy of Usina Moreno (located in Planalto-SP)

and was collected from the first storage tank situated after the distillation unit. The

industrial process carried at Usina Moreno involves the use of the excess molasses

from sugar production together with sugarcane juice to produce ethanol.

Vinasse composition was determined by BioAgri Laboratories (registration

number 278887/2011-0) and is presented in Table 16.

Experiments were also carried in a synthetic medium, known as Clostridium

acetobutylicum medium (CAB), largely used in cultivation of Clostridia. CAB medium

contains, per liter: 4.0 g yeast extract, 1.0 g tryptone, 1.5 g K2HPO4, 0.5 g

asparagine, 1 ml of 0.2%(v/v) resazurin, 0.1 g MgSO4.7H2O, 0.1 g MnSO4.H2O, 15

mh FeSO4.7H2O, 0.1 g NaCl, 10 g sucrose. pH was adjusted to 7,0 with KOH.

Fermentations were compared to a not fermented medium. The results

present in tables are difference between the fermented medium and the not

fermented medium (negative results means consumption of the respective VFA).

2.4 High Performance Liquid Chromatography (HPLC) and Ethanol

quantification.


Chromatography (HPLC). Before injection the samples (2 ml) were centrifuged and

filtered (Milipore 0,2µm).

The HPLC equipment was an Shimadzu Liquid Chromatograph equipped with

a Aminex® HPX-87H 300 x 7,8mm (Bio-Rad) column and a refractive index detector

(RID-10A). The column was kept at 60oC and a 5mM H2SO4 at 0,6 ml/min was used

as mobile phase. The compounds quantified by this method are glucose, fructose,

54

succinate, lactate, formate, acetate, propionate and butyrate. All chemicals used

were of analytical grade.

Because the retention time of butyrate and ethanol are very similar, it was

impossible to differentiate them by HPLC. The method used for determining ethanol

content was based on the oxidation of ethanol to acetic acid by reaction with

potassium dichromate in an acidic medium. The solution acquires a green color

proportional to the ethanol concentration in the sample, enabling the reading on the

spectrophotometer at 600 nm. The standard solution of potassium dichromate (1 L)

consisted of the following components: 500 ml of distilled H2O, 325 mL concentrated

H2SO4 and 33.678 g of potassium dichromate.

2.5 Gas Analysis

Gas was collected by inserting a graduated syringe through the flange-type

butyl rubber septum (Figure 8). Cultures degassed daily were considered free of H2

partial pressure, different from those degassed twice a week (4th and 7th days).

Figure 8 – The use of a graduated syringe in the quantification of the gas produced during fermentation

55

The gas from some generations (those that were analyzed for VFAs) had the

produced gas purified for hydrogen content estimation. Purification was carried by an

adaptation of a widely used technique that involves the pulverization of the biogas a

column containing a 10% NaOH solution. This system was used because carbon

dioxide and H2S reacts instantly reacts with NaOH, but hydrogen do not. The tower

used was made of glass and was filled 50% of its volume with different sized glass

beads in order to increase gas contact time with the basic solution. Gas was injected

at approximately 2 ml/s through a porous stone. Hydrogen content was estimated by

dividing the volume obtained after and before purification.


3.1 Vinasse composition

The vinasse used in these experiments was analyzed for its composition and is

presented in Table 16.

It can be noticed the presence of important ions for the production of

biohydrogen, such as iron, manganese, magnesium and phosphorus. Moreover, the

low content of nitrogen indicates that microbial growth will be greatly limited unless it

is added to the medium.

Regarding this, total nitrogen content in the medium supplemented with

molasses and sugarcane juice was determined by the Kjeldahl method. In molasses

supplemented medium, nitrogen content was approx. 73 mg/L while in sugarcane it

was approx. 27mg/L.

56

Table 17 – Complete composition of the vinasse used during the experiments was carried by BioAgri Laboratory.

Parameter mg/L

Iron 41,8

Manganese 3,7

Lead <0,1

Cadmium <0,1

Mercury <0,00035

Arsenium <0,1

pH 4,52

Nitrate <10

Total Nitrogen (Kjeldahl) 2,15

Sodium 20,1

Calcium 791

Potassium 2386

Magnesium 203

Sulphate 1700

Total Phosphorus 104,9

DBO 8358

DQO 29600

3.2 Strains metabolism analysis

Metabolic behavior in terms of VFAs and hydrogen production of each

strain/consortia is depicted in this section. Due to the complexity of the metabolism

of hydrogen producers and the use consortia a metabolic analysis is very intricate.

Moreover, the synergetic effects of hydrogen partial pressure and carbon source on

hydrogen producer metabolisms further increase this complexity.

57

3.2.1 ATCC 8260

According to the described methods, VFAs and gas production analysis were

carried and the results are shown in Table 18. It can be noticed a great difference in

the profile of VFAs when a complex substrate was used in comparison with pure

sucrose. Moreover, different sources of carbon resulted in different profile of VFAs.

The negative concentrations found indicates the consumption of these metabolites in

comparison to the non-fermented medium.

Table 18 – Metabolic products of the cultivation of ATCC 8260 in vinasse medium containing different carbon sources. Results include cultivation allowing and avoiding H2 partial pressure. VFAs concentration is shown in

g.L-‐1. Results are the average of 5 analyses.

With H2 partial pressure

Strain Carbon source Succinic Lactic Formic Acetic Propionic Butyric Ethanol

Hydrogen (L/L)

ATCC 8260

CAB 0 8,070 0 0.089 0 0.230 0 <0,40

Juice 0 0 0 1,196 0,074 4,485 0 1,35±0,33

Molasses 0 0 0 0,352 0,241 3,982 0 0,73±0,17

Sucrose 0,285 0 2,695 0,14 -0,707 3,211 0 1,84±0,34

Without H2 partial pressure

Strain Carbon source Succinic Lactic Formic Acetic Propionic Butyric Ethanol

Hydrogen (L/L)

ATCC 8260 Juice 0 0,299 0 3,446 0,624 7,594 0 1,70±0,3

Molasses 0 0 0 -‐1,032 0,988 4,242 0 1,04±0,12 Sucrose 0 0 0 1,263 -‐0,41 5,798 0 2,77±0,21

It can be noticed that ATCC8260 is mainly an acetate-butyrate producer,

except when cultured in the synthetic CAB medium. In medium containing sucrose

and avoiding H2 pressure, only these two VFAs were produced, while in CAB

medium high amounts of lactic acid was produced. Sucrose was the carbon source

that gave best hydrogen and VFAs yields.

In terms of hydrogen production, cultivation of ATCC 8260 in artificial medium

resulted in very low amounts when compared to vinasse-based medium.

Considerable improvement in H2 production was noticed on fermentations avoiding

H2 pressure, which was expected.

58

3.2.2 ATCC 27021

The strain ATCC 27021 presented the best results for biohydrogen production

in the preliminary experiments (Chapter 2). A great effort was made in order to keep

that productivity but the strain showed to be very sensible and hard to work with (at

frequent time intervals the culture showed no gas production). This was also noted

by partners that started working with this strain at Blaise Pascal University. Because

this would result in difficulties at manipulation in industrial scale, this strain was no

longer used.

3.2.3 C6

Volatile fatty acids and hydrogen production by this strain is showed in Table

19. This strain is a potential ethanol producer, which was also observed in the

experiments described in Chapter 2.

Table 19 -‐ Metabolic products of the cultivation of the strain C6 in vinasse medium containing different carbon sources. Results include cultivation allowing and avoiding H2 partial pressure. VFAs concentration is shown in

g.L-‐1. Results are the average of 5 analyses.


Strain Carbon source Succinic Lactic Formic Acetic Propionic Butyric Ethanol Hydrogen (L/L)

C6

CAB 0 7,932 0 0 0 0 0 0,48±0,08

Juice 0 0 0 2,677 0,172 3,612 2,71 0,99±0,32

Molasses 0 0 0,172 1,231 0,065 2,895 1,77 0,95±0,23 Sucrose 0,716 0 0,906 -‐0,292 -‐0,146 4,895 1,07 1,36±0,22



C6 Juice 0 0 0,335 2,375 0,513 6,557 2,21 1,83±0,55

Molasses 0 0 0,816 -‐1,052 -‐0,3875 3,099 0,32 1,99±0,45 Sucrose 0,129 0 0,466 -‐0,274 -‐0,392 5,448 1,11 1,68±0,23

High hydrogen production was achieved using molasses as carbon source and

avoiding hydrogen partial pressure, situation which resulted in lower ethanol

production. When sugarcane juice was used as carbon source a great amount of

59

butyric acid was produced, but the presence of other VFAs would result in laborious

purification process.

As observed for ATCC 8260, large amounts of lactic acid were produced when

CAB medium was used. Accompanied by this, very low amounts of hydrogen were

produced.

3.2.4 VINA

The metabolic analysis (Table 20) of the consortium VINA showed a great

proportional effect of hydrogen partial pressure in ethanol production. Because this

consortium was originated from the vinasse itself, the ethanol production observed

was expected.

The use of molasses and sugarcane juice also caused changes in metabolism,

probably due to variations in the consortium composition caused by the different

composition of such complex substrates. Pure sucrose was the best carbon source

for hydrogen production, followed by molasses.

It is interesting to note that when the synthetic medium was used, again a

completely different profile of VFAs was noted. At the same time, and as observed in

the previous strains and consortia, lower amount of hydrogen and great amounts of

lactic acid was produced when compared to vinasse-based medium.

60

Table 20 -‐ Metabolic products of the cultivation of the consortium VINA in vinasse medium

containing different carbon sources. Results include cultivation allowing and avoiding H2 partial

pressure. VFAs concentration is shown in g.L-‐1. Results are the average of 5 analyses.



VINA

CAB 0,352 7,724 1,371 0 0 0 0 <0,40 Juice 0 0 0,058 2,803 0,123 2,999 3,19 0,78±0,32

Molasses 0 0 1,793 1,932 0,508 2,908 1,99 1,13±0,31 Sucrose 1,428 0 0,507 -‐0,801 -‐0,288 3,821 2,04 1,66±0,35



VINA Juice 0 1,837 0 -‐1,144 3,135 1,729 2,99 0,51±0,18

Molasses 0 0 0 0,95 -‐0,1125 1,324 0,52 1,84±0,26 Sucrose 0,499 0 0,671 3,219 -‐0,208 1,75 2,33 2,58±0,41

When cultured in vinasse medium the consortium LPB AH3 presented a very

high production of butyric acid for all substrates tested. Sucrose was the best carbon

source for hydrogen production in vinasse medium, with yields slight higher than

those of sugarcane juice (Table 21).

The use of molasses resulted in very low hydrogen yield but high amount of

butyric acid. The highest amount of butyric acid (10 g.L-1) among all strains

evaluated was produced by this consortium (in sugarcane juice supplemented

medium).

61

Table 21 -‐ Metabolic products of the cultivation of the consortium LPB AH3 in vinasse medium containing different carbon sources. Results include cultivation allowing and avoiding H2 partial pressure. VFAs

concentration is shown in g.L-‐1. Results are the average of 5 analyses.



LPB AH3

CAB 0,105 0 0,627 2,150 0 1,5105 0.3450 0,58±0,06 Juice 0 0 0 2,167 0,627 6,073 1,78 0,83±0,26

Molasses 0 0 0 -‐1,046 0,267 4,127 1,06 <0,40 Sucrose 0 0 0 3,113 0,576 5,238 0,99 1,37±0,18



LPB AH3

Juice 0 0 0 -‐1,236 1,243 10,088 2,21 1,04±0,14

Molasses 0 0 0 0,917 1,3435 7,13 1,09 0,60±0,19 Sucrose 0 0 0 3,069 0,798 7,896 1,02 1,63±0,10

Because the amount of metabolites produced is greater than the available

substrate for fermentation, we can conclude that this consortium is capable of using

other components from vinasse, sugarcane juice and molasses as carbon source.

3.2.5 LPB AH1

The consortium LPB AH1 presented a high capacity to produce biohydrogen in

vinasse medium, especially when sucrose or sugarcane juice was used as carbon

source (Table 21). When CAB medium was used, about half of the biohydrogen

production achieved in vinasse medium supplemented with sugarcane juice and

sucrose was achieved.

From Table 22 it can be noticed that in vinasse medium supplemented with

sugarcane juice the effect of H2 partial pressure in H2 production was minimum. This

is very interesting considering industrial application because facilitates process

handling. On the other hand, the profile of VFAs under and avoiding H2 pressure was

very different, resulting in propionic acid accumulation in the first condition. The

effect of the synthetic medium in the consortia development can also be noticed by

the production of formic acid instead of propionate.

62




Strain Carbon source Succinic Lactic Formic Acetic Propionic Butyric Ethanol Hydrogen

(L/L)

LPB AH1

CAB 0.14 0 0.285 1.324 0 1.216 0 0,83±0,1

Juice 0 0 0 0,599 1,321 6,793 0 2,03±0,31

Molasses 0 0 0,067 1,157 0,118 4,322 0,2 1,15±0,24 Sucrose 0 0 0,163 -‐0,581 -‐0,2 6,995 0 2,08±0,19



(L/L)

LPB AH1 Juice 0 0 0,185 3,525 0,408 7,642 0 2,25±0,29

Molasses 0,05 0 0 -‐2,085 -‐0,4025 4,421 0 1,97±0,26

Sucrose 0 0 1,393 1,049 -‐0,451 4,824 0 2,94±0,31

3.2.6 LPB AH2

Metabolic analysis of the consortium LPB AH2 showed a great potential for

biohydrogen production in molasses and sugarcane juice supplemented media

(Table 23). The use of molasses as carbon source together with the maintenance of

a low H2 partial pressure environment resulted in the exclusive production of butyrate

as VFA.

Higher H2 production was observed in fermentations carried under reduced H2

pressure, except in sucrose supplemented medium where no statistical difference

was noted, which is interesting because facilitates the management of the process in

an industrial scale.

63





(L/L)

LPB AH2

CAB 0 0 0,06 2,12 0 1,028 0 0,99±0,08

Juice 0 4,197 0 2,956 0,311 6,313 0 1,74±0,42

Molasses 0 0 0 3,783 0,383 6,396 0 1,45±0,29 Sucrose 1,17 0 0,876 1,333 -‐0,398 7,044 0 2,29±0,42



(L/L)

LPB AH2 Juice 0 0 0 -‐2,62 0,478 8,000 0 2,16±0,35

Molasses 0 0 0 -‐1,793 -‐0,1355 6,067 0 2,17±0,25

Sucrose 2,749 0 0,518 4,709 -‐0,72 6,809 0 2,37±0,20

3.2.7 LPB AH4

The VFAs profile generated by the fermentation of vinasse based medium with

the consortium LPB AH4 is presented in Table 24. It can be noticed that a mix of

acetic, propionic, butyric and ethanol (and formic acid in sucrose supplemented

medium) was produced. It is interesting to observe that in synthetic CAB medium

lactate was produced, which was not noted in vinasse-based medium.

In terms of hydrogen production, we can note that vinasse medium resulted in

higher yields, which is consistent with the theory depicted in Chapter 1 (more

reduced products results in less hydrogen yield).

64





(L/L)

LPB AH4

CAB 0,4395 8,76 0 0 0 1,0612 0,487 0,55±0,1

Juice 0 0 0 1,574 0,045 2,649 1,612 0,81±0,20

Molasses 0 0 0 0,671 1,173 2,248 0,83 0,82±0,26 Sucrose 0 0 0,639 -‐0,439 -‐0,53 2,59 0,99 1,25±0,26



(L/L)

LPB AH4 Juice 0 0 0 1,06 1,269 4,466 1,1 0,93±0,28

Molasses 0 0 1,505 1,394 -‐0,2125 2,138 0,88 1,07±0,23

Sucrose 0,649 0 0,564 -‐0,052 0,042 6,317 0,41 1,34±0,30

3.2.8 LPB AH5

The consortium LPB AH5 didn’t presented capacity to use molasses and

sugarcane juice as carbon sources for growth. Because sucrose was consumed both

in synthetic (CAB) and vinasse media it is possible that some constituent(s) of

molasses and juice is (are) toxic to this consortium. Even in sucrose based media

the amount of VFAs and hydrogen produced was too low.

65





LPB AH5

CAB 0 0,083 0,573 0,451 0 0,677 0 <0,40

Juice 0 0 0 0 0 0 0 0

Molasses 0 0 0 0 0 0 0 0 Sucrose 0 0 0,088 2,058 2,125 0,658 1,43 0,52±0,05



LPB AH5 Juice 0 0 0 0 0 0 0 0

Molasses 0,363 0 0 -‐0,659 0,287 0,02 0 <0,40

Sucrose 0 0 0 3,192 2,579 1,469 0,54 0,54±0,10

*not possible to determine (above limit of detection of the method used)

3.2.9 LPB AH6

When cultivated in the media supplemented with sucrose, the consortium LPB

AH6 presented a high hydrogen production. In vinasse based medium this condition

was achieved since a low H2 pressure was kept.

The consortium LPB AH6 was the only consortium to presented adaptation to

molasses but no adaptation to sugarcane juice, which was not expected because

molasses is usually more toxic to some microorganisms.

66





LPB AH6

CAB 0 9,591 0,99 0 0 0 0 0,61±0,08

Juice 0 0 0 0 0 0 0 0

Molasses 0 0 0,26 1,363 1,041 2,57 1,05 1,15±0,32

Sucrose 1,006 0 0,606 0,662 -‐0,53 2,717 0,76 1,47±0,35



LPB AH6

Juice 0 0 0 0 0 0 0 0

Molasses 0 0 0 -‐2,103 2,2065 2,004 0,88 1,58±0,27

Sucrose 1,321 0 0,604 0,301 0,797 3,291 0,82 2,31±0,38

3.2.10 LPB AH7

The consortium LPB AH7 showed a behavior similar to the observed for the

consortium LPB AH5: hydrogen and VFAs production was observed only in those

media where pure sucrose was used as carbon source.





LPB AH7

CAB 0 9,030 0 0 0 0 0 0,62±0,11

Juice 0 0 0 0 0 0 0 0

Molasses 0 0 0 0 0 0 0 0

Sucrose 0 0 0 3,169 3,756 2,054 0 0,37±0,10



LPB AH7

Juice 0 0 0 0 0 0 0 0

Molasses 0 0 0 0 0 0 0 0

Sucrose 1,321 0 0,823 0 2,954 0,185 0 0,51±0,08

67

3.3 Conclusions

Because our objective was to develop an economic feasible process of

biohydrogen production with the possibility to take advantage of the VFAs produced,

the selection of strains was carried considering the capacity to produce biohydrogen

in vinasse medium supplemented with complex carbon sources (molasses and

sugarcane juice) and the profile of VFAs produced.

The consortium LPB AH2 presented the highest H2 production capacity in

vinasse medium with molasses (2.17 LH2/Lmedium). At this condition, only butyrate was

produced, at considerable amount (6.1 g.L-1), which is interesting and facilitates its

recovery.

In terms of butyric acid production, the consortium LPB AH3 achieved the

highest value (10 g.L-1), but because of considerable amounts of propionate and

ethanol production, the H2 productivity was low in comparison to others.

In sugarcane juice supplemented medium the consortium LPB AH1 presented

the best results. Hydrogen production reached 2.25 LH2/Lmedium, accompanied by

considerable amounts of acetate and butyrate production (3.5 and 7.6 g.L-1,

respectively), which is relevant in coupling to methane or solvent production.

Two consortia presented considerable ethanol production: C6 and VINA, both

in sugarcane supplemented medium and in environment with high H2 partial

pressure. The first one achieved a production of 2.71 g.L-1 while the other reached

3.19 g.L-1. This is approx. 40% of the ethanol that is produced by yeast fermentation

through traditional fermentation. Ethanol associated to hydrogen production in

vinasse medium may be interesting due to the possibility of this ethanol recuperation

be facilitated since it is quite possible that the bioH2 facilities are installed coupled to

the ethanol plant (more specifically the distillation unit). On the other hand this

technology competes with traditional ethanol production due to use of sugarcane

juice as substrate and can probably be considered if someday greater restrictions for

vinasse disposal are imposed.

It might be also considered the possibility to produce large amounts of lactic

acid using the synthetic CAB medium (further studies should be carried on this

theme). At the same time, those consortia that produced lactic acid in the synthetic

medium but didn’t on vinasse medium indicates that changes in process conditions

68

(in vinasse composition, for example) can result in the generation of undesirable

products instead of hydrogen.

Based on these observations, the consortium LPB AH2 was chosen for

biohydrogen and VFAs production in vinasse medium supplemented with molasses

and LPB AH1 in vinasse medium with sugarcane juice.

69

CHAPTER 4

Optimization of culture

conditions of the consortia LPB

AH2 and LPB AH1 cultivated in

vinasse-based medium for

biohydrogen and VFAs

production under anaerobic

conditions.

70

1 Introduction Based on the metabolic analysis of each strain/consortia described in chapter

3, 2 strains were selected as potential biohydrogen and VFAs producers: LPB AH2

and LPB AH1. Before process scaling up an optimization step was carried in order to

achieve highest biohydrogen production.

Many factors that fall under the topic of bioprocess parameters have been

studied including type of organism/organisms, pH, substrate loading (OLR – organic

loading rate), type of reactor/growth conditions (batch, sequencing batch,

continuous; CSTR, UASB, etc.), type of substrate (pure carbohydrate, various waste

streams), media composition, ions availability, etc. Several approaches that can be

considered to increase hydrogen yields in the dark fermentation will be discussed in

this chapter.

The yield of hydrogen during dark fermentation is severely affected by

the partial pressure of the product. At high H2 partial pressures a metabolic shift to

production of more reduced products, like lactate or ethanol occurs, decreasing the

yield of H2. The formation of relatively reduced organic molecules is an integral part

of all dark fermentations and some of these molecules (e.g. acetate) can inhibit H2

production if allowed to accumulate (12). Metabolic engineering of hydrogen

producing microorganisms to minimize production of other more reduced products by

blocking their biosynthetic pathways is an alternative to provide higher H2 yields (13,

14, 15). Gas sparging has also been found to be a useful technique to reduce

hydrogen partial pressure in the liquid phase for enhancement of its yield (32) but

results in difficulties in hydrogen purification.

In terms of carbon source, only acids are produced when carbon source is

limited in the medium (75). Unlike carbon-limited cultures, solvents are produced by

cultures grown in phosphate- or sulfate-limited media. pH is also an important factor,

as high fermentation rates lead to strong acidification due to the production of

organic acids. This can affect both product distribution and biomass production.

Higher hydrogen yields will most probably be achieved by limiting cell growth through

nutrient limitations, thereby enhancing catabolic processes but high cell densities are

needed to maximize hydrogen production rates.

71

The balance of the medium to reach this optimal point is crucial in process

development. The determination of the composition of complex media for industrial

applications plays, thus, an important role in development and maintenance of an

industrial H2 process. Yu et al (57b) reported, for example, that the production of

acetate was inhibited by Zn and Cu; but production of propionate and hydrogen was

favored at low concentrations of Zn (up to 80 mg l-1) and Cu (up to 40 mg l-1). Other

studies indicate that nitrogen, phosphorous and iron are the most important essential

nutrients for hydrogen gas production (59). Magnesium ion is also an important

cofactor that activates almost 10 enzymes including hexokinase,

phosphofructokinase and phosphoglycerate kinase during glycolysis process (66).

Hawkes et al (67) reviewed the media composition for hydrogen production.

They found that apart from N and P source, only K, Mg and Fe are common in all

recipes in analyzed. A 20-fold variation in the amount of Fe added with respect to

hexose concentration was also observed. One or more workers did not add one or

more of the elements Ni, Ca, B, Mo, Zn, Co, Cu, Mn or I. Hydrogen production

described in the literature showed large variation and most of the time no relation is

established with inorganic nutrients consumption. More information on minimum

amounts of these nutrients for continuous operation is needed.

The use of hyper-thermophiles and extreme temperatures in hydrogen

production represents some gains in terms of hydrogen yields, since at increased

temperatures hydrogen production becomes more exergonic (17). Therefore,

extreme- and hyper-thermophiles show a better resistance to high hydrogen partial

pressures (18). Another advantage of fermentations at extreme temperatures is that

the process is less sensitive to contaminations by hydrogen consumers. The major

problems are (i) to achieve an economical relation between the energy used in order

to heat and maintain the reactor at high temperatures and the H2 production, and (ii)

that extreme thermophiles anaerobic bacteria usually grow to low densities resulting

in low production rates.

In this chapter, the optimization of culture parameters was conducted

considering the fact that biohydrogen technology faces economical drawbacks. It

was described that some micronutrients play an important role in biohydrogen

production and could had been considered. But since the main goal is to develop an

72

economic and simple-to-handle process, the smaller the changes made in medium

composition, the better for process economics. In this context only pH and the

carbon/nitrogen ratio were optimized.

pH and carbon are probably the most important factors to be regulated in

anaerobic digestion processes. They play a critical role in governing the metabolic

pathways of microbial H2 production [77] and the composition of the microbial

community.

Process optimization was carried by using the Response Surface Methodology

(RSM), a widely used technique to model processes in which the response of

interest (in this case, biohydrogen production) is influenced by several variables (pH

and C/N). Because fist-order models won’t be enough, a central composite design

(CCD) was chosen in order to estimate with more accuracy the mathematical

behavior of biohydrogen production.


2.1 Medium Composition and Culture Conditions


volume of 6 ml, sealed with autoclavable Bakelite lids with rubber stoppers and

incubated in a shaker at 37ºC and 30 rpm. The cultures were maintained at these

conditions for 1 week and then inoculated in a new medium. 1 ml of culture was,

then, inoculated in 5 ml of medium. Each new culture will be called “generation”.

Medium pH was adjusted with 1N KOH. Anaerobic environment and medium

was carried according to the Balch technique. Bicarbonate was added at 85ºC and

Cysteine-HCl at 65ºC as reducing agents to lower the redox potential of medium.

Biohydrogen and VFAs production by the consortium LPB AH2 was carried

using vinasse medium supplemented with sugarcane molasses, while the

consortium LPB AH1 was cultivated in vinasse medium supplemented with

sugarcane juice. Vinasse, molasses and sugarcane juice used in these experiments

were the same used in the previous chapter.

73



Chromatography (HPLC). Before injection the samples (2 ml) were centrifuged for 10

min at 104 g and filtered (Milipore 0.2µm).

The HPLC equipment was an Shimadzu Liquid Chromatograph equipped with


(RID-10A). The column was kept at 60oC and a 5mM H2SO4 at 0.6 ml/min was used


succinate, lactate, formate, acetate, propionate and butyrate. All chemicals used

were of analytical grade. Ethanol quantification was carried as described in chapter

3.

2.3 Gas Measurement and Analysis

Before analysis, 7 successive cultivations were made in order to achieve a

balanced microbial community (resulting in a theoretical stability of the process).

Hydrogen partial pressure was minimized by daily degassing. Total gas production

(Lgas/Lmedium) was considered as the sum of the gas produced and quantified daily

divided by the volume of medium.

Gas analysis was carried twice a week, more precisely in the 4th and 7th day of

culture. Gas was collected by inserting a graduated syringe through the flange-type

butyl rubber septum. The gas collected in the 4th day was purified for hydrogen

content estimation, as follows.

Since it was noted in chapter 2 that there is a direct relation between gas

production and hydrogen content in the gas phase, the optimal conditions was

considered as the one that resulted in higher (bio)gas production.

2.4 Strains

The strains used in this experiments were those selected based on the results

of chapter 3. The consortium LPB AH2 was chosen for biohydrogen and VFAs

production in vinasse medium supplemented with molasses, while the consortium

74

LPB AH1 was chosen for biohydrogen and VFAs production in vinasse medium

supplemented with sugarcane juice.

2.5 Optimization and data analysis

Optimization was carried using a statistical tool called “Essential Experimental

Design”, version 2.213. An inscribed central composite design with 2 factors at 3

levels and 3 center points was used for each strain. The response used for

optimization was total gas produced (in Lgas/Lmedium) since it was noted in chapter 2

that there is a direct relation between gas production and hydrogen content in the

gas phase. The statistical plan is showed in Table 28. Table 30 shows the values

assigned to each level.

75

Table 29 – Statistical plan used for the optimization of conditions for biohydrogen and VFAs production by the chosen consortia.

Exp # Carbon Source (g/L) pH

1 -1 -1 2 0 0 3 1 -1 4 -1 1 5 1 1 6 0 0 7 0 -1,414 8 0 1,414 9 -1,414 0

10 0 0 11 1,414 0

Table 30 – Values of pH and carbon source assigned to each level of the optimization plan.

Carbon Source (g/L) Level -1,414 -1 0 1 1,414 Value 7,93 10 15 20 22,07

pH

Level -1,414 -1 0 1 1,414 Value 4,88 5,5 7 8,5 9,12


3.1 Consortium LPB AH1 cultivated in vinasse medium supplemented

with sugarcane juice.

The experimental results for gas production by the consortium LPB AH1 are

presented in Table 31. The effect of pH and carbon source concentration on

hydrogen production are represented in the 3-D and Contour plots presented in

Figure 9. A maximum production of biogas of respectively 8,29Lgas/Lmedium occured at

pH 7,0 and 12g/L carbon source.

RE�

�

� � � o� � TS � m� � � 2� DU: � 7� 3°: e� � � N°� P� � � � t � � 7o3°P� 3°e � 3N� � � : e2: U3°7r � � � � � � S � 7e� � U� � : e� °3°: e2� � � � : U� °e � 3: � 3N� �23� 3°23°� � o� r : � � o� 72� � � 1: U� : D3°r °b� 3°: eu�

Exp # Carbon Source (g/L) pH Gas (L/Lmedium) 1 -1 -1 7,38 2 0 0 8,25 3 1 -1 2,04 4 -1 1 7,46 5 1 1 3,00 6 0 0 8,29 7 0 -1,414 2,88 8 0 1,414 2,89 9 -1,414 0 6,33

10 0 0 8,27 11 1,414 0 2,25

�

�

77

Figure 9 – Graphical 3-‐D and contour displays of the achieved results for optimization of gas production by LPB AH1 consortium cultivated in vinasse medium supplemented with sugarcane juice.

The best mathematical model that fit satisfactory to the results is a full quadratic

model (Table 32), presenting a R² higher than 0,91. This means that it is possible to

predict hydrogen production by the consortium LPB AH1 grown in terms of pH and

substrate concentration.

Low coefficient of variation approx. 20% and standard error (1,095) were

observed, which was impressive since higher variation was expected because of the

complex composition of the medium (sugarcane juice and vinasse). VIF value under

5 indicates the inexistence of multicollinearity among the regressors (Table 30).

Table 32 -‐ The equation of the full quadratic model that fit best to the results achieved in this optimization is presented. Coefficient values, standard errors, 95% interval of confidence and T student are also shown.

Gás_(ml) = b0 + b1*Fonte de Carbono (g/L) + b2*pH*pH + b3*Fonte de Carbono (g/L)*Fonte de Carbono (g/L) + b4*Fonte de Carbono (g/L)*pH +

b5*pH

P value Std Error -95% 95% t Stat VIF

b0 8,270 4,65041E-05 0,632 6,645 9,894 13,09

b1 -1,946 0,00400 0,387 -2,941 -0,952 -5,029 1,000

b2 -2,347 0,00379 0,461 -3,531 -1,163 -5,095 1,095

b3 -1,644 0,01606 0,461 -2,829 -0,460 -3,569 1,095

b4 0,220 0,704 0,547 -1,187 1,627 0,402 1,000

b5 0,132 0,747 0,387 -0,863 1,127 0,341 1,000

The Durbin-Watson statistic test was carried but was inconclusive for the

detection of autocorrelation in the residuals (dL<d<dU; 0.758<1.094<1.604; interval

of confidence = 95%). A first order autocorrelation (Pearson’s r) value of 0.358 was

observed and indicates a weak positive autocorrelation between residuals. This is

important because high positive autocorrelation means biased estimated coefficients

in the mathematical model and suggests that other variables should be included.

The ANOVA analysis presented in Table 33 showed a low percentage of

residuals, indicating that the predicted responses are close to the obtained ones.

78

The F test confirms that the model is valid in a confidence interval of 99%

(Fsignif<confidence interval).

Table 33 – The ANOVA analysis showed low content of residuals and indicates that the full quadratic equation proposed is valid.

ANOVA Source SS SS% MS F F Signif df

Regression 67,36 92 13,47 11,24 0,00946 5 Residual 5,991 8 1,198

5

LOF Error 5,990 8 (100) 1,997 4991,4544 0,000200 3 Pure Error 0,000800 0 (0) 0,000400

2

Total 73,35 100

10

Table 32 presents the VFAs produced in each condition of optimization. It can

be observed that conditions where low quantities of biogas was produced was

related with high amounts of lactic acid production, excepted the one of pH 7 and

7,93 g.L-1 substrate, in which low amount of gas is probably related to low content of

fermentable carbon. It was also noticed that conditions of high pH and/or high carbon

source concentration favored the development of lactic acid bacteria; in these

conditions formic acid was also produced.

79

Table 34 – Volatile fatty acids production of the consortium LPB AH1 during optimization. Substrate, succinic, lactic, formic, acetic, propionic and butyric acids are showed in g.L-‐1.

pH Substrate Succinic Lactic Formic Acetic Propionic Butyric Gas (L/Lmedium)

4,88 15,00 0,000 7,994 3,175 0,969 -‐0,576 1,331 2,88

5,5 10,00 0,000 0,000 0,000 2,430 0,046 6,123 7,38

5,5 20,00 0,000 8,323 3,218 0,772 -‐0,371 2,321 2,04

7 7,93 0,000 0,000 0,000 1,995 -‐0,187 5,150 2,88

7 15,00 0,000 0,000 0,000 2,280 0,146 7,638 8,27

7 22,07 0,000 9,257 3,564 0,952 -‐0,009 2,589 2,25

8,5 20,00 0,000 12,998 4,946 1,050 -‐0,456 1,916 3,00

8,5 10,00 0,000 0,615 0,000 0,000 -‐0,218 6,728 7,46

9,12 15,00 0,000 10,737 4,146 1,343 -‐0,150 2,874 2,89

3.2 Consortium LPB AH2 cultivated in vinasse medium supplemented

with sugarcane molasses.

The experimental results for gas production by the consortium LPB AH2 are

presented in Table 35. The lowest production observed was 1,83 Lgas/Lmedium when

the consortia was cultivated at the lowest pH, while the highest (7,67 Lgas/Lmedium) was

achieved at the central point (pH 7,0 and 15g.L-1 substrate). Figure 10 presents the

effects of the conditions on biohydrogen production.

I c �

�

� � � o� � TH� m� � � 2� DU: � 7� 3°: e� � � N°� P� � � � t � � 7o3°P� 3°e � 3N� � � : e2: U3°7r � � � � � � S � 7e� � U� � : e� °3°: e2� � � � : U� °e � 3: � 3N� �23� 3°23°� � o� r : � � o� 72� � � 1: U� : D3°r °b� 3°: eu�

Exp # Carbon Source (g/L) pH Gas (Lgas/Lmedium) 1 -1 -1 5,88 2 0 0 7,61 3 1 -1 3,88 4 -1 1 5,92 5 1 1 6,33 6 0 0 7,56 7 0 -1,414 1,83 8 0 1,414 3,72 9 -1,414 0 5,67

10 0 0 7,67 11 1,414 0 6,44

�

�

� ° 7U� � Sc � i � � U� DN°� � o� Ti � � � e� � � : e3: 7U� � °2Do� t 2� : 1� 3N� � � � N°� P� � � U� 27o32� 1: U� : D3°r °b� 3°: e� : 1� � 2�

DU: � 7� 3°: e� � t � � � � � � C� � : e2: U3°7r � � 7o3°P� 3� � � °e� P°e� 22� � r � � °7r � 27DDo� r � e3� � � s °3N� 27 � U� � e� �

r : o� 22� 2u�

81

The best mathematical model that fit satisfactory to the profile of the results

achieved is a full quadratic model (Table 36) with R² of approx. 0,90. It means that it

is possible to predict hydrogen production by the consortium LPB AH2 grown in

vinasse medium supplemented with sugarcane molasses.

Table 36 – The equation of the full quadratic model that fit best to the results achieved in this optimization is presented. Coefficient values, standard errors, 95% interval of confidence and T student are also shown.

Gas_(ml) = b0 + b1*pH*pH + b2*pH + b3*substrate (g/L)*substrate (g/L) + b4*substrate (g/L)*pH + b5*substrate (g/L)

P value Std Error -95% 95% t Stat VIF

b0 7,615 1,58788E-05 0,467 6,413 8,816 16,29 b1 -2,148 0,00148 0,341 -3,024 -1,273 -6,306 1,095 b2 0,645 0,07381 0,286 -0,09032 1,381 2,255 1,000 b3 -0,508 0,196 0,341 -1,384 0,368 -1,491 1,095 b4 0,603 0,197 0,405 -0,438 1,643 1,489 1,000

b5 -0,06266 0,835 0,286 -0,798 0,673 -0,219 1,000

As observed for the LPB AH1 consortium, a low coefficient of variation and a

low standard error were achieved (14% and 0,809, respectively). The Durbin-Watson

statistic test was again inconclusive for the detection of autocorrelation in the

residuals (dL<d<dU; 0,758<1,057<1,604; interval of confidence = 95%). A first order

autocorrelation (Pearson’s r) value of 0,347 was observed and indicates a weak

positive autocorrelation between residuals.

The ANOVA analysis presented in Table 37 showed a low percentage of

residuals. The F test showed that the model is valid in a confidence higher than 98%.

Table 37 – The ANOVA analysis showed low content of residuals and indicates that the full quadratic equation proposed is valid.

ANOVA Source SS SS% MS F F Signif df

Regression 30,96 90 6,193 9,452 0,01384 5 Residual 3,276 10 0,655

5

LOF Error 3,270 10 (100) 1,090 360,3381 0,00277 3 Pure Error 0,00605 0 (0) 0,00303

2

Total 34,24 100 10

82

In terms of VFAs production (Table 38), it can be observed that in extreme

conditions of pH and carbon source lactate production was observed, especially in

high pH (above 8,5). It was possible to note that in fermentations that lactic acid was

produced in great quantity low amount of gas was released. In most fermentations

only butyric acid was observed, which is in accordance to results achieved in chapter

3.

Table 38 – Volatile fatty acids production of the consortium LPB AH2 during optimization. The concentration of the carbon source, succinic, lactic, formic, acetic, propionic and butyric acids are showed in g.L-‐1.

pH Carbon Source Succinic Lactic Formic Acetic Propionic Butyric Gas (L/Lmedium)

4,88 15 0,000 2,017 0,000 -0,493 1,390 6,794 2,88

5,5 10,00 0,000 0,000 0,000 -0,450 -0,877 1,850 7,38

5,5 20 0,000 0,000 0,000 -0,643 -0,755 3,675 2,04

7 7,93 0,000 0,364 0,000 0,092 -0,756 1,538 6,33

7 15,00 0,000 0,000 0,000 -0,846 -0,293 6,065 7,61

7 22,07 0,000 5,817 0,000 -0,655 -0,628 4,296 2,25

8,5 20 0,000 8,095 0,000 -0,948 -0,696 3,403 3,00

8,5 10,00 0,000 0,368 0,000 0,659 0,514 6,289 7,46

9,12 15,00 0,000 7,936 0,000 -0,012 -0,558 0,836 2,89

4 Conclusions The optimization of the conditions of culture resulted in higher biohydrogen

production close to the central points for both consortia. In terms of pH it was

expected since the experiments described in chapters 3 and 4 were conducted at pH

7,0. Anyway it was not observed flourishing of hydrogen-producers resistant to

extreme pHs. The methodology chosen and the design proposed can predict through

a mathematical model how biohydrogen is produced in relation to pH and carbon

source concentration.

It was not expected that such a uniform behavior could be achieved. Because

the consortia are composed by more than one microorganism its adaptation to

different conditions is facilitated, which is confirmed considering that hydrogen

83

production was observed even at very low or high pHs. It would be of great value if a

considerable biohydrogen production was achieved in low pH because vinasse’s

natural pH is usually close to 5.

84

CHAPTER 5

Scaling up: bioreactor cultivation

of consortia under optimized

conditions for biohydrogen and

VFAs production in vinasse-

based medium

85

1 Introduction

Dark fermentative biohydrogen processes is found to be most often performed

in closed vessels. Closed batch mode is generally used as the first step to examine

physical factors (type of substrate, carbon content, temperature, gas pressure)

affecting the process (78) as a first step in process development. Generally H2

production and growth kinetics are successfully investigated through this technique.

Regarding industrial application (large scale operations), biohydrogen

processes are expected to work in continuous mode in most cases. CSTR

(continuous stirred tank reactor) is the most commonly studied, where the hydraulic

retention time (HTR) is the parameters of greatest influence. They are also preferred

in terms of ease of operation. The concentrations of volatile fatty acids in the digester

are proportional to the organic loading rate (OLR) and to HRT.

A variety of organic load rates (OLR) have been tested and although the results

are highly variable given the different substrates used. It is obvious that high

substrate concentrations are to be preferred from an operational standpoint since

they potentially lead to high volumetric production rates. The effect of OLR, at least

in mixed cultures, on hydrogen yields is somewhat contradictory with no easy

explanation for the disparity in the results. In pure culture fermentations hydrogen

yields are favored at low carbon concentrations whereas hydrogen productivity is

favored at high carbon concentrations. Recent studies with mixed cultures also

generally support this idea, although the relationship seems more complex (61). Kim

et al. (68) reported that short HRT would favor hydrogen production as methanogens require

more than approx. 3 days HRT before they were washed out from a CSTR reactor.

Low HTR generally results in low operation costs and is used to eliminate

methane producers. On the other hand the efficiency of the process is reduced

(biomass growth and hydrogen production is limited, especially in CSTR) and it is

observed loss of fermentable sugar in the wastewater. The optimal HTR for each

process must be evaluated because it changes according to substrate and inoculum.

Generally CSTR generates higher H2 productivity but with lower yields when

compared to batch mode.

To overcome the low biomass production (and consequently low hydrogen

productivity) in continuous operating reactors, the use of immobilized cells or

I E�

�

methods to allow formation of granules or flocs is being considered. Examples are

the use of fixed-bed (79) and membrane reactors (80).

In batch reactors the highest yield described was achieved using the

thermophile Caldicellulosiruptor owenensis (4.0 molH2/molglucose) (82) while non-

thermophile strains can reach up to 3,10 molH2/molglucose (83). The highest evolution

rate of 35 mmol L-1 h-1 was described in a culture of Enterobacter cloacae II BT-08

grown in sucrose-rich synthetic medium in batch mode (YH2/S = 6,0) (84), less than

half the amount achieved with the same strain cultured in continuous mode (77

mmol L-1 h-1) (YH2/S not described) (85).

This chapter describes the scaling up of the proposed biohydrogen process

using vinasse as medium and the optimized conditions as described in previous

chapter to a bench reactor operated in batch mode. The objective is to evaluate the

metabolism of both consortia selected to obtain valuable information for a future

development of continuous operation. An economical discussion is also carried

based on the results achieved.

�

' � � S� E� � � � N� � U � S� I � R�

cnu � d� yd1� � � � � � y� � H� � � � � � y1� � � H�

The experiments were carried out in a 2L bioreactor, with working volume of

1,5L, adapted for anaerobic cultivation (Figure 11). Batch fermentations were

maintained at 37ºC and without agitation during 5 days.

87

Figure 11 – 2L Bioreactor used in scaled up production of biohydrogen and VFAs by the consortium LPB AH2 (cultivated in vinasse medium supplemented with sugarcane molasses) and LPB AH1 (cultivated in vinasse

medium supplemented with sugarcane juice).

Medium pH was adjusted with 1N KOH. Anaerobic environment and medium

was carried according to the Balch technique. The reactor was autoclaved and a

anaerobic environment was created by CO2 injection in the headspace. Bicarbonate

was added when the medium temperature reached 85ºC and Cysteine-HCl at 65ºC

as reducing agents to lower the redox potential of medium. The bioreactor was then

kept overnight under CO2 environment prior inoculation.

Carbon source concentration and pH were set according to the results

achieved in the previous chapter. Biohydrogen and VFAs production by the

consortium LPB AH2 was carried in vinasse medium supplemented with 15g/L

sugarcane molasses (based on obrix), while cultivation of the consortium LPB AH1

was carried in vinasse medium supplemented with 12 g/L sugarcane juice (based on obrix). The initial pH for each strain was 7.0.

Inoculum production was carried through serial inoculations. A 6 mL daily

degassed culture was inoculated in 50 ml new medium. After 5 days of culture and

daily degassing, this culture was used as inoculum for a 300ml culture, which was

then inoculated in the bioreactor.

88

2.2 Vinasse

The vinasse used in bioreactor scale was the same used in the previous

chapter. Its composition is presented in Table 16, chapter 3.



Chromatography (HPLC). Samples were withdrawed daily, centrifuged for 10 min at

104 g and filtered (Milipore 0,2µm) before injection.

The HPLC equipment was a Shimadzu Liquid Chromatograph equipped with


(RID-10A). The column was kept at 60oC and a 5mM H2SO4 at 0.6 ml/min was used


succinate, lactate, formate, acetate, propionate and butyrate. All chemical used were

of analytical grade.

2.4 Gas Measurement and analysis

Gas measurement was carried using an inverted beaker (Figure 12)

connected by a rubber hose to the bioreactor gas exit. Gas production was

considered equal to the volume of displaced water.

89

Figure 12 – The system of gas measurement (foreground) adapted to the bioreactor (background).

At the end of the fermentation, 40ml of the accumulated gas was sampled and

analyzed. Gas analysis was carried at the Institute for Technology Development

(Instituto de Tecnologia para o Desenvolvimento – LACTEC) in a Thermo Gas

Chromatographer equipped with the following analytical columns: Petrocol DH150

(50mx0,25mm), DC 200 (1,8m) and Porapak-N (2,0m x 1/8’’), which were placed in

by-pass series flow path of gas chromatograph system. The columns were

connected to a TCD detector (block temperature: 120°C, transducer temperature:

120°C, filament temperature: 190°C). This system allowed the measurement of

oxygen (O2), nitrogen (N2), carbon dioxide (CO2) and methane (CH4). Hydrogen (H2)

content was then considered as the amount to reach 100%.

2.5 Other Analysis

Biomass was quantified daily by centrifuging 10ml samples at 16500g and

drying at 60oC until constant weight. Total carbohydrate was quantified daily by the

Phenol-Sulfuric method. pH was monitored daily in a digital pHmeter.

5c �

�

G � � RT SR��

t nu � � � g� 1 � � � � � � � � � � � H� 21 � d� y� � � � � � � � 1� � � y 1� H� � � � � � g� y� � �

� � H 1y� d� � � � � � � u�Biohydrogen and VFAs fermentation process using the consortium LPB AH1

was carried using vinasse and sugarcane juice as medium at the conditions

described in Material and Methods. Initial sugar content in the fermentation medium

was equal to 11.48g.L-1, being completely exhausted by the end of fermentation.

Biomass production was equal to 0.25g.L-1.

The VFAs and biomass production profile during the 5 days of fermentation

are showed in Figure 13, while gas production, carbohydrate consumption and pH

variation is showed in Figures 13 and 14.

�� ° 7U� � ST� i � � 7UP� 2� : 1� � °: r � 22� � e� � � � � 2� DU: � 7� 3°: e� � 7U°e � 3N� � � 7o3°P� 3°: e� : 1� 3N� � � : e2: U3°7r � � � � � � S � °e� P°e� 22� �

r � � °7r � 27DDo� r � e3� � � s °3N� 27 � U� � e� � d7°� � u��

5S�

�

�� ° 7U� � SA� i � � °: � 2� DU: � 7� 3°: eh� 27� 23U� 3� � � : e27r D3°: e� � e� � D � P� U°� 3°: e� � 7U°e � 1� Ur � e3� 3°: e� : 1� P°e� 22� �

27DDo� r � e3� � � s °3N� 27 � U� � e� � d7°� � � � t � 3N� � � : e2: U3°7r � � � � � � Su��

It was noticed a high production of VFAs, mainly butyrate and lactate, on the

first 24 hours, which was accompanied by a high rate of sugar and propionic acid

consumption, pH decrease and biogas production. More than 60% of the gas that

has been produced until the end of fermentation was produced in this first 24hours

time interval.

On the second day of fermentation, it was noticed the consumption of the

carbon source and some of the VFAs produced in the first day (lactic, acetic and

formic acids) resulting in propionic acid, gas and biomass production. This might be

a consequence of flourishing of different microorganism(s) (apparently propionic

bacteria) from those present in the first 24 hours of fermentation.

Because no considerable differences in VFAs profile was noticed after the

second day of fermentation, we can state that the consumption of such VFAs was

directed towards hydrogen and biomass production (which is confirmed by

comparing Figures 10 and 11).

These results indicate that a continuous fermentation process for biohydrogen

and butyric acid by the consortia LPB AH1 can be carried with a hydraulic retention

time of 48 hours (since butyric acid is a desired product). In this process very low

concentration of lactic, acetic and propionic acid would be found and more than 90%

of the carbohydrate would be consumed. This proposed reduction of fermentation

time for a continuous process was carried by Zhang et al. (2006) (81) and resulted in

92

a reduction of the diversity of microbial community associated with an elimination of

propionate production without affecting the existence of dominant pure cultures.

On the other hand, conducting fermentation during only 24 hours will result in

higher H2 yield and productivity but a mixture of VFAs that will demand a more

laborious downstream prior purification, if this pure specific acid(s) is (are) desired.

3.1.1 Metabolic analysis According to the results presented in figures 13 and 14, a µmax of 0,15 d-1

was achieved. The substrate consumption rate in the first 48 hours was equal to 4,07

g.L-1.d-1. Approximately 2,2% of the substrate was used in biomass production (YX/S),

while almost 39% was used for butyrate production (44,6% considering every acid

produced – YVFAs/S). This means that 53,2% of the consumed substrate was probably

used in CO2 production (YCO2/S) and cellular maintenance (Ym/S).

At the end of the fermentation, 8,08Lgas/Lmedium was produced, an amount very

close to the one predicted during optimization in chapter 4 (8,85 Lgas/Lmedium). The

biogas composition is presented in Table 39. The presence of very low quantities of

oxygen indicates insignificant contamination of the gas before analysis and shows

that an anaerobic condition for cultivation was successfully achieved. Table 39 – Composition of the biogas produced during the fermentation by the consortium LPB AH1.

Biogas components Content (%)

Nitrogen 2,13

Carbon dioxide 66,2

Oxygen 0,62

Methane 0,00

Hydrogen* 31,05

*Hydrogen content was estimated by the amount to reach 100%

Considering the carbon dioxide percentage on the biogas and considering that

it is behaving as an ideal gas, we can estimate the amount of substrate used in CO2

as follows:

If P.V=n.R.T where P=1atm, V=(66,25%*8,08Lgas/Lmedium), R= 0,082057

atm.L.mol-1.K-1 and T (K)=37oC+273,15oC, the value for nCO2 is 0,21 mol/L (9,25

5T�

�

gCO2/L). If all the 53,2% of the cited consumed substrate was destined for CO2

production, the amount of the carbon dioxide produced should be:

CO2 = 0,532*11,48gsubstrate/L*1,54gCO2/gsucrose = 9,41gCO2/L

which means that approx. 98% of the 53,2% consumed substrate was

destined for CO2 production. The conclusion is, finally, that YCO2/S = 52% while Ym/S

is approx. 1,0%. A general representation of the destination of the substrate

consumed by the consortium LPB AH1 is showed in Figure 15.

�� ° 7U� � SH� i � � � DU� 2� e3� 3°: e� : 1� 3N� � � � 23°e� 3°: e� : 1� 3N� � 27� 23U� 3� � °e� 3� Ur 2� : 1� � � � 2h� � °: r � 22� � e� � � � C� DU: � 7� 3°: e�

� e� � � � oo7o� U� r � °e3� e� e� � u�

GAÁA' B� EI � � N� VEI � T� S� I N� VI S� NS� � � � N� BR� R�The maximum productivity of hydrogen was achieved considering the first 24

hours of fermentation, reaching 61,5 mlH2.L-1.h-1 (which means 2,75 mmolH2.L-1.h-1).

This productivity can be considered low, since it is quietly normal to find

productivities of 5-20 mmolH2.L-1.h-1 in the literature.

On the other hand, a yield of 7,14 molH2.molsucrose-1 was achieved, which is as

high as 89,25% of the theoretical maximum yield. This is very high and is achieved

more frequently using thermophiles.

This opposite behavior of achieving high molH2.molCsource-1 and low

productivities is well described in the literature. In order to achieve high conversion

rates, generally long times are needed, which consequently results in low

productivity. As example, a very high productivity of 50mmol.L-1.h-1 was achieved but

with yields as low as 0,09molH2.molglucose-1 by culturing Bacteroides fragilis in glucose

94

rich medium (86). Probably higher productivities (with lower conversion rate) could

be achieved if hourly analysis were carried during the exponential H2 production

phase.

Considering the hydrogen content in the biogas, the inferior and superior

calorific powers (ICP and SCP) were calculated and estimated as 9050 kcal.kg-1 and

10730 kcal.kg-1, respectively. In comparison to a methane rich biogas (65% CH4 –

ICP = 7735 kcal.kg-1 and 8612 kcal.kg-1), the calorific power presented by the

hydrogen rich biogas is superior (17% higher).

At this point, a reflexion might be carried in terms of the feasibility of usage of

hydrogen-rich biogas as heat source instead of methane-rich biogas. The difference

in terms of calorific power is considerable and indicates that hydrogen-rich biogas is

better, but the complexity of the technology to produce it is greatly superior to the

one to produce methane. This suggests that to became feasible, considering that

hydrogen content in the biogas could not be greatly increased, the proposed

technology might consider the purification of hydrogen (adding value to the final

product).

3.2 Biohydrogen and VFAs production in bioreactor scale by the

consortium LPB AH2

Biohydrogen and VFAs fermentation process using the consortium LPB AH2

was carried using vinasse and sugarcane juice as medium, according to the results

achieved in previous chapters. Carbohydrate concentration at the beginning of the

fermentation was quantified as 13.42 g/L and the initial pH was 7,0.

Almost 50% of the carbon source was consumed in the first 24 hours and was

exhausted in the last day of fermentation (Figure 16). Biomass production was equal

to 0.65g/L and final pH was 5.15.

5H�

�

�� ° 7U� � SE� i � � °: � 2� DU: � 7� 3°: eh� 27� 23U� 3� � � : e27r D3°: e� � e� � D � P� U°� 3°: e� � 7U°e � 1� Ur � e3� 3°: e� : 1� P°e� 22� �

27DDo� r � e3� � � s °3N� 27 � U� � e� � r : o� 22� 2� � t � 3N� � � : e2: U3°7r � � � � � � Cu��

�� ° 7U� � SR� i � � 7UP� 2� : 1� � °: r � 22� � e� � � � � 2� DU: � 7� 3°: e� � 7U°e � 3N� � � 7o3°P� 3°: e� : 1� 3N� � � : e2: U3°7r � � � � � � S � °e� P°e� 22� �

r � � °7r � 27DDo� r � e3� � � s °3N� 27 � U� � e� � r : o� 22� 2u��

The HPLC analysis of the samples withdrawed daily showed a complex

behavior of VFAs production (Figure 17). In the first 24 hours butyric, acetic and

lactic acids were produced in large amounts, while low quantities of formic and

propionic acid were identified. This was accompanied by great carbohydrate uptake

(6.21g.L-1), a great biogas production (58% of the gas that would be produced by the

end of the fermentation) and a pH drop to 5.9. In the second day of fermentation, the

VFAs were consumed and gas production lowered, which is possibly an effect of

96

consortia composition variation. During the third day of fermentation little changes

was noticed, but after that butyric, acetic and low amounts of propionic acids were

observed together with carbohydrate consumption. Biomass production was greatly

increased in this time interval (4th-6th day of fermentation) and gas production rate

was kept relatively constant.

3.2.1 Metabolic analysis According to the results presented in figures 16 and 17, a µmax of 0.375 d-1

was achieved. The maximum substrate consumption rate (first 24 hours) was equal

to 6.21 g.L-1.d-1 (YX/S). About 4.9% of the substrate was used in biomass production

(YX/S), while almost 41.5% was used for VFAs production (YVFAs/S). This means that

53.6% of the consumed substrate was probably used in CO2 production (YCO2/S) and

cellular maintenance (Ym/S).

In comparison to the optimization prediction of gas production in chapter 4

(7.76 Lgas/Lmedium), 6.41Lgas/Lmedium was produced at the end of the fermentation in

the bioreactor. This difference illustrate the expected variation of biogas (and

consequently biohydrogen) production caused by the use of complex medium and a

consortium of microorganisms.

The biogas composition is presented in Table 40. The presence of very low

quantities of oxygen indicates an insignificant contamination of the gas before

analysis and shows that an anaerobic condition for cultivation was successfully

achieved. Table 40 – Composition of the biogas produced during the fermentation by the consortium LPB AH2.

Biogas components Content (%) Nitrogen 3.93

Carbon dioxide 62.4 Oxygen 0.97 Methane 0.00

Hydrogen* 32.7 *Hydrogen content was estimated by the amount to reach 100%

The mass balance to determine YCO2/S and Ym/s was carried as described in

section 3.2.1. If P.V=n.R.T where P=1atm, V=(62.4%*6.41Lgas/Lmedium), R= 0.082057

atm.L.mol-1.K-1 and T (K)=37oC+273.15oC, the value for nCO2 is 0.157 mol/L (6.91

5R�

�

gCO2/L). If all the 53.6% of the cited consumed substrate was destined for CO2

production, the amount of the carbon dioxide produced should be:

CO2 = 0.532*13.42gsubstrate/L*1.54gCO2/gsucrose = 10.99gCO2/L

which means that approx. 82% of the 53.6% consumed substrate was

destined for CO2 production. The conclusion is, finally, that YCO2/S = 43.9% while Ym/S

is approx. 9.7%. A general representation of the destination of the substrate

consumed by the consortium LPB AH1 is showed in Figure 18.

�� ° 7U� � SI � i � � � DU� 2� e3� 3°: e� : 1� 3N� � � � 23°e� 3°: e� : 1� 3N� � 27� 23U� 3� � °e� 3� Ur 2� : 1� � � � 2h� � °: r � 22� � e� � � � C� DU: � 7� 3°: e�

� e� � � � oo7o� U� r � °e3� e� e� � u��

GA' A' B� EI � � N� VEI � T� S� I N� VI S� NS� � � � N� BR� R�The maximum productivity of hydrogen was achieved considering the first 24

hours of fermentation, reaching 55.07 mlH2.L-1.h-1 (which means 2.46 mmolH2.L-1.h-1)

with a yield of 3.25 molH2.molsucrose-1 (41% of the theoretical maximum yield). The

highest yield was achieved considering 2 days of fermentation (3.66 molH2.molsucrose-

1), but the productivity was as low as 35.2 mlH2.L-1.h-1.

Considering the hydrogen content in the biogas, the inferior and superior

calorific powers (ICP and SCP) were calculated and estimated as 9483 kcal.kg-1 and

11248 kcal.kg-1, respectively. In comparison to a methane rich biogas (65% CH4 –

ICP = 7735 kcal.kg-1 and 8612 kcal.kg-1), the calorific power presented by the

hydrogen rich biogas is superior (22.6% higher).

�

98

4. Conclusion

According to the results achieved in the experimentations described in this

chapter showed the feasibility to produce hydrogen in the conditions described in a

rector scale. The metabolic analysis by daily analysis of biogas production and VFAs

provided important information to the development of a continuous process, which is

more feasible to the proposed technology.

Through material and energy balance it was possible to estimate how the

energy of the substrate is distributed during the fermentation. Moreover, growth

indicators were calculated are of great value in further development of this process.

Considering the possibility of associating this technology to a biogas or

solvent production process (for biogas production, VFAs works as substrates), it is

important to note that there is a production of considerable amounts of VFAs. The

process developed with the consortium LPB AH1 has potential for butanol production

since butyrate concentration in the broth is much higher than other VFAs.

In terms of how the hydrogen produced can be used, a deep analysis might

be carried. The first point is that the proposed technology depends on sugarcane

molasses or juice, which are used in ethanol production. In order to be considered

promising (and to be transferred to the industry) the proposed technology should

provide substantial economic gains. Thus, 3 scenarios of usage of hydrogen by the

ethanol industry are proposed:

i) Use in direct heat generation: Ethanol industries use sugarcane bagasse in

boilers to produce heat. Sugarcane bagasse (with 20% water) PCS is 3641

kcal/kg, almost 3 times lower than the biogas generated by using vinasse

supplemented with sugarcane juice.

A ethanol plant that produced 1000 m³ of ethanol/day uses approx. 12000

tons of sugarcane, resulting in approx. 1800tons of sugarcane bagasse, which

are capable to generate 6,55*109 kcal. In this scenario, the increase in the

energy generated by using biogas as a complementary source of energy will

be insignificant (hydrogen-rich biogas can produce up to 8,5*106 kcal/day

considering the production rate and biogas composition achieved using LPB

AH1 consortium) .

99

This suggests that at the current level of development the use of hydrogen for

direct heat production is unfeasible.

ii) Use in fuel cells: Ethanol industries generally produce more energy than they

use, selling the surplus electricity to local electric companies. Anyway,

hydrogen could be used to enhance this energy production.

Considering a 1,2 kW proton exchange membrane fuel cell that uses

hydrogen with purity of 99.99%, at a consumption rate of 18.5 L/min (31), 20

m³ of vinasse based medium was needed to produce enough hydrogen to

operate when considering fermentation of vinasse and molasses with the

consortium LPB AH2 at the conditions described in this chapter. When

considering the use of the consortium LPB AH1 (vinasse supplemented with

sugarcane juice) the volume needed is of approx. 19 m³.

Considering the realistic daily production of 1000m³ ethanol by an ethanol

plant, which means the daily generation of 12 thousand m³ of vinasse, approx.

720kW of energy could be produced. The average price of the KWh in 2012 in

Brazil was R$0,333 (U$0,17), which means that monthly approx. U$3600 in

energy could be produced using hydrogen in fuel cell, which is very low.

iii) Purification of hydrogen: the price of pure analytic hydrogen is approx. U$

56,50/m³ (White Martins, Brazil). By the proposed technology, fermenting 1 m³

of vinasse generates daily approx. 1,5 m³ of pure hydrogen (rate achieved

using LPB AH1 consortium). Considering an ethanol plant generating 12000

m³ of vinasse, approx. U$ 1 million dollar/day can be obtained by selling pure

H2.

100

General Conclusion The scientific advances for the reuse of industrial wastewaters for the

production of compounds promote the recovery of the energy and nutrients that were

lost in wastewater treatments. But in most cases the compounds produced from this

feedstock are not feasible to be used in food/feed or as pharmaceuticals, making

biofuels production an important alternative.

Among the biofuels feasible to be produced through biological methods (oils,

biodiesel, CH4, H2) hydrogen is the one with higher energy density and is told to be

the fuel of the future. Its production through fermentation of agroindustrial wastes

(liquids and solids) issues zero extra carbon to the atmosphere, being considered

thus an eco-friendly source of energy. Moreover, the flexibility of hydrogen as a final

product (use in direct combustion for heat production, to produce electricity and/or in

chemical industries) is another big advantage.

In present, fermentation technologies to produce hydrogen are in a basic

level of development and face economical drawbacks, but are gaining importance in

the last years (more than 20 thousand papers were published in the last 10 years).

Considerable advances in this topic will probably be achieved soon. It is unanimous

that the use of agroindustrial residues (solid and liquid) is the main alternative to

overcome this economic dilemma. In this context, it is important to consider that the

feedstock might be produced in large amounts. If it presents great potential but is

generated in small quantities, its use for biohydrogen production is unworthy.

In Brazil, vinasse is the industrial liquid residue produced in most quantity: in

2013/2014 it is expected to be produced almost 26 billion liters of ethanol, which

means approx. 550 billion liters of vinasse. Despite presenting benefits when used

as fertilizer, the issues that are being observed as consequence of its disposal will

certainly result in increasing government (environment) restrictions to protect the

environment. Furthermore most ethanol industries generate vinasse in excess and

gives not rational destination to it.

The production of biohydrogen from vinasse is interesting because of the

possibility to use it as source of energy within the industry in an integrated process.

In this work a process for the production of biohydrogen fermenting vinasse with

anaerobic bacteria was developed. The study covered the search and screening of

101

microorganisms, and also the optimization and production in bioreactor scale,

presenting interesting results. This work represents an important first step of the

development of a process to be used industrially.

102

Future works The present work opens the possibility of innumerous studies to be carried in

the field. Exploration of new microorganisms should be carried constant since the

biodiversity seems to be an inexhaustible source of improvement. The

characterization of the consortia might give valuable information and should be

carried.

Since the bioreactor experiments were carried only in batch mode, it is of

great interest to carry fermentations in continuous and feed-batch mode. By

controlling the hydraulic retention time in continuous fermentations it will be probably

possible to select those microorganisms with higher hydrogen productivity. Different

fermenters should also be tested: membrane reactors, UASB (upflow anaerobic

sludge blanket), and others.

The effluent of the hydrogen fermentation can be evaluated for the production

of methane in a coupled process. It can also be evaluated for its potential as fertilizer

for sugarcane, which would be of great interest.

103

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Publications

• Patent: Soccol, C.R.; Sydney, E. B.; Larroche C. "Processo para Produção

de Hidrogênio e Ácidos Graxos Voláteis", PI1005215-1, 2010;

• Sydney, E.B. ; da Silva, T.E. ; Tokarski, A. ; Novak, A.C. ; de Carvalho, J.C. ;

Woiciecohwski, A.L. ; Larroche, C. ; Soccol, C.R. . Screening of microalgae

with potential for biodiesel production and nutrient removal from treated

domestic sewage. Applied Energy, v. 88, p. 3291-3294, 2011.

• Angelis, S. ; Novak, A. C. ; Sydney, E. B. ; Carvalho, J. C. ; Pandey, A. ;

Noseda, M. D. ; Tholozan, J. L. ; Lorquin, J. ; Soccol, C. R. . Co-Culture of

Microalgae, Cyanobacteria, and Macromycetes for Exopolysaccharides

Production: Process Preliminary Optimization and Partial Characterization.

Applied Biochemistry and Biotechnology, v. 167, p. 1092, 2012.

• Patent: Soccol, C.R.; Novak, A.C. ; SOCCOL, A. T. ; SYDNEY, E. B. ; de

ANGELIS, S. “Processo para produção de exopolissacarídeos, biomassa e

extratos antioxidantes”, BR 1020120046318, 2012.

• Sarma, S. J.; Brar, S. K.; Sydney, E. B.; Le Bihan, Y.; Buelna, G.; Soccol, C.

R. Microbial hydrogen production by bioconversion of crude glycerol: A

review. International Journal of Hydrogen Energy, v. 37, p. 6473-6490, 2012.

• Dos Santos, J. D.; Lopes da Silva, A. L.; Da Luz Costa, J.; Scheidt, G. N.;

Novak, A. C.; Sydney, E. B. ; Soccol, C. R. Development of a vinasse

nutritive solution for hydroponics. Journal of Environmental Management, v.

114, p. 8-12, 2013.

• Book Chapter: Eduardo Bittencourt Sydney, Alessandra Cristine Novak,

Julio Cesar de Carvalho, Carlos Ricardo Soccol. Respirometric Balance and

Carbon Fixation of Industrially Important Algae. Biofuels from Algae, 1st

Edition. Elsevier. 2013.

• Book Chapter: Carlos José Dalmas Neto, Eduardo Bittencourt Sydney,

Ricardo Assmann, DolivarCoraucci Neto,Carlos Ricardo Soccol. Production of

115

Biofuels from Algal Biomass by Fast Pyrolysis. Biofuels from Algae, 1st

Edition. Elsevier. 2013.

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